Novel gene

ABSTRACT

An isolated nucleic acid molecule mapping to chromosome 16q24.3 and comprising the nucleotide sequence set forth in SEQ ID Numbers: 1 or 3.

RELATED APPLICATIONS

This application is a divisional patent application which claims thebenefit of the filing date of U.S. patent application Ser. No.10/10/470,700, filed Dec. 20, 2002, which claims benefit to PCTInternational Patent Application Serial No. PCT/AU01/00729, filed Jul.29, 2003, the disclosures of which are incorporated herein by referencein their entirety.

TECHNICAL FIELD

The present invention relates to a novel gene which has been identifiedat the distal tip of the long arm of chromosome 16 at 16q24.3. The BNO1gene encodes a polypeptide that forms part of a ubiquitin-ligase complexinvolved in targeting proteins by ubiquitination for degradation by theproteasome. In view of the realisation that BNO1 is involved inubiquitination and protein degradation, the invention is also concernedwith the therapy of disorders associated with this process, such ascancer (in particular breast and prostate carcinoma),immune/inflammatory disease and neurological disease. In addition, theinvention is concerned with the diagnosis of disorders associated withubiquitination and the screening of drugs for therapeutic interventionin these disorders.

BACKGROUND ART

The development of human carcinomas has been shown to arise from theaccumulation of genetic changes involving both positive regulators ofcell function (oncogenes) and negative regulators (tumour suppressorgenes). For a normal somatic cell to evolve into a metastatic tumour itrequires changes at the cellular level, such as immortalisation, loss ofcontact inhibition and invasive growth capacity, and changes at thetissue level, such as evasion of host immune responses and growthrestraints imposed by surrounding cells, and the formation of a bloodsupply for the growing tumour.

Molecular genetic studies of colorectal carcinoma have providedsubstantial evidence that the generation of malignancy requires thesequential accumulation of a number of genetic changes within the sameepithelial stem cell of the colon. For a normal colonic epithelial cellto become a benign adenoma, progress to intermediate and late adenomas,and finally become a malignant cell, inactivating mutations in tumoursuppressor genes and activating mutations in proto-oncogenes arerequired (Fearon and Vogelstein,1990).

The employment of a number of techniques, such as loss of heterozygosity(LOH), comparative genomic hybridisation (CGH) and cytogenetic studiesof cancerous tissue, all of which exploit chromosomal abnormalitiesassociated with the affected cell, has aided in the identification of anumber of tumour suppressor genes and oncogenes associated with a rangeof tumour types.

In one aspect, studies of cancers such as retinoblastoma and coloncarcinoma have supported the model that LOH is a specific event in thepathogenesis of cancer and has provided a mechanism in which to identifythe cancer causing genes. This model is further highlighted in VonHippel-Lindau (VHL) syndrome, a rare disorder that predisposesindividuals to a variety of tumours including clear cell carcinomas ofthe kidneys and islet cell tumours of the pancreas. Both sporadic andinherited cases of the syndrome show LOH for the short arm of chromosome3 and somatic translocations involving 3p in sporadic tumours, andgenetic linkage to the same region in affected families has also beenobserved. The VHL tumour suppressor gene has since been identified fromthis region of chromosome 3 and mutations in it have been detected in100% of patients who carry a clinical diagnosis of VHL disease. Inaddition, the VHL gene is inactivated in approximately 50-80% of themore common sporadic form of renal clear cell carcinoma.

The genetic determinants involved in breast cancer are not as welldefined as that of colon cancer due in part to the histological stagesof breast cancer development being less well characterised. However, aswith colon carcinoma, it is believed that a number of genes need tobecome involved in a stepwise progression during breast tumourigenesis.

Certain women appear to be at an increased risk of developing breastcancer. Genetic linkage analysis has shown that 5 to 10% of all breastcancers are due to at least two autosomal dominant susceptibility genes.Generally, women carrying a mutation in a susceptibility gene developbreast cancer at a younger age compared to the general population, oftenhave bilateral breast tumours, and are at an increased risk ofdeveloping cancers in other organs, particularly carcinoma of the ovary.

Genetic linkage analysis on families showing a high incidence ofearly-onset breast cancer (before the age of 46) was successful inmapping the first susceptibility gene, BRCA1, to chromosome 17q21 (Hallet al., 1990). Subsequent to this, the BRCA2 gene was mapped tochromosome 13q12-q13 (Wooster et al., 1994) with this gene conferring ahigher incidence of male breast cancer and a lower incidence of ovariancancer when compared to BRCA1.

Both BRCA1 and BRCA2 have since been cloned (Miki et al., 1994; Woosteret al., 1995) and numerous mutations have been identified in these genesin susceptible individuals with familial cases of breast cancer.

Additional inherited breast cancer syndromes exist, however they arerare. Inherited mutations in the TP53 gene have been identified inindividuals with Li-Fraumeni syndrome, a familial cancer resulting inepithelial neoplasms occurring at multiple sites including the breast.Similarly, germline mutations in the MMAC1/PTEN gene involved inCowden's disease and the ataxia telangiectasia (AT) gene have been shownto confer an increased risk of developing breast cancer, among otherclinical manifestations, but together account for only a smallpercentage of families with an inherited predisposition to breastcancer.

Somatic mutations in the TP53 gene have been shown to occur in a highpercentage of individuals with sporadic breast cancer. However, althoughLOH has been observed at the BRCA1 and BRCA2 loci at a frequency of 30to 40% in sporadic cases (Cleton-Jansen et al., 1995; Saito et al.,1993), there is virtually no sign of somatic mutations in the retainedallele of these two genes in sporadic cancers (Futreal et al., 1994;Miki et al., 1996). Recent data suggests that DNA methylation of thepromoter sequence of these genes may be an important mechanism ofdown-regulation. The use of both restriction fragment lengthpolymorphisms and small tandem repeat polymorphic markers has identifiednumerous regions of allelic imbalance in breast cancer suggesting thepresence of additional genes, which may be implicated in breast cancer.Data compiled from more than 30 studies reveals the loss of DNA from atleast 11 chromosome arms at a frequency of more than 25%, with regionssuch as 16q and 17p affected in more than 50% of tumours (Devilee andCornelisse, 1994; Brenner and Aldaz, 1995). However only some of theseregions are known to harbour tumour suppressor genes shown to be mutatedin individuals with both sporadic (TP53 and RB genes) and familial(TP53, RB, BRCA1, and BRCA2 genes) forms of breast cancer.

Cytogenetic studies have implicated loss of the long arm of chromosome16 as an early event in breast carcinogenesis since it is found intumours with few or no other cytogenetic abnormalities. Alterations inchromosome 1 and 16 have also been seen in several cases of ductalcarcinoma in situ (DCIS), the preinvasive stage of ductal breastcarcinoma. In addition, LOH studies on DCIS samples identified loss of16q markers in 29 to 89% of the cases tested (Chen et al., 1996; Radfordet al., 1995). In addition, examination of tumours from other tissuetypes have indicated that 16q LOH is also frequently seen in prostate,liver, ovarian and primitive neuroectodermal carcinomas. Together, thesefindings suggest the presence of a gene mapping to the long arm ofchromosome 16 that is critically involved in the early development of alarge proportion of breast cancers as well as cancers from other tissuetypes, but to date no such gene has been identified.

DISCLOSURE OF THE INVENTION

The present invention provides an isolated nucleic acid molecule mappingto chromosome 16q24.3 comprising the nucleotide sequence set forth inSEQ ID Numbers: 1 or 3.

It also provides an isolated nucleic acid molecule comprising thenucleotide sequence set forth in SEQ ID Numbers: 1 or 3, or a fragmentthereof, which encodes a polypeptide that forms part of aubiquitin-ligase complex involved in targeting proteins byubiquitination for degradation by the proteasome.

The invention also encompasses an isolated nucleic acid molecule that isat least 70% identical to a DNA molecule consisting of the nucleotidesequence set forth in SEQ ID Numbers: 1 or 3 and which encodes apolypeptide that forms part of a ubiquitin-ligase complex involved intargeting proteins by ubiquitination for degradation by the proteasome.

Such variants will have preferably at least about 85%, and mostpreferably at least about 95% sequence identity to the nucleotidesequence encoding BNO1. A particular aspect of the invention encompassesa variant of SEQ ID Numbers: 1 or 3 which has at least about 70%, morepreferably at least about 85%, and most preferably at least about 95%sequence identity to SEQ ID Numbers: 1 or 3. Any one of thepolynucleotide variants described above can encode an amino acidsequence, which contains at least one functional or structuralcharacteristic of BNO1.

Typically, sequence identity is calculated using the BLASTN algorithmwith the BLOSSUM62 default matrix.

The invention also encompasses an isolated nucleic acid molecule thatencodes a polypeptide that forms part of a ubiquitin-ligase complexinvolved in protein degradation through ubiquitination, and whichhybridizes under stringent conditions with a DNA molecule consisting ofthe nucleotide sequence set forth in SEQ ID Numbers: 1 or 3.

Under stringent conditions, hybridization will most preferably occur at42° C. in 750 mM NaCl, 75 mM trisodium citrate, 2% SDS, 50% formamide,1×Denhart's, 10% (w/v) dextran sulphate and 100 μg/ml denatured salmonsperm DNA. Useful variations on these conditions will be readilyapparent to those skilled in the art. The washing steps which followhybridization most preferably occur at 65° C. in 15 mM NaCl, 1.5 mMtrisodium citrate, and 1% SDS. Additional variations on these conditionswill be readily apparent to those skilled in the art

The invention also provides an isolated nucleic acid molecule whichencodes a polypeptide having the amino acid sequence set forth in SEQ IDNumbers: 2 or 4.

Still further, the invention encompasses an isolated nucleic acidmolecule wherein the encoded amino acid sequence has at least 70%,preferably 85%, and most preferably 95%, sequence identity to thesequence set forth in SEQ ID Numbers: 2 or 4.

Preferably, sequence identity is determined using the BLASTP algorithmwith the BLOSSUM62 default matrix.

In a further aspect, there is provided an isolated nucleic acid moleculecomprising exons 1 to 9 or exons 1, 2, 2.5, and 3 to 9 identified in thenucleotide sequences set forth in SEQ ID Numbers: 1 and 3 respectively.

Still further, there is provided an isolated nucleic acid moleculeconsisting of the nucleotide sequence set forth in SEQ ID Numbers: 1 or3.

In a still further aspect, there is provided an isolated nucleic acidmolecule consisting of the nucleotide sequence set forth in SEQ ID NO: 1from base 4 to base 1,621 or set forth in SEQ ID NO: 3 from base 4 tobase 1,708.

In a further aspect, the invention provides an isolated gene comprisingthe nucleotide sequence set forth in SEQ ID Numbers: 1 or 3 and BNO1control elements.

Preferably, the BNO1 control elements are those which mediate expressionin breast, prostate, liver and ovarian tissue.

The nucleotide sequences of the present invention can be engineeredusing methods accepted in the art so as to alter BNO1-encoding sequencesfor a variety of purposes. These include, but are not limited to,modification of the cloning, processing, and/or expression of the geneproduct. PCR reassembly of gene fragments and the use of syntheticoligonucleotides allow the engineering of BNO1 nucleotide sequences. Forexample, oligonucleotide-mediated site-directed mutagenesis canintroduce mutations that create new restriction sites, alterglycosylation patterns and produce splice variants etc.

As a result of the degeneracy of the genetic code, a number ofpolynucleotide sequences encoding BNO1, some that may have minimalsimilarity to the polynucleotide sequences of any known and naturallyoccurring gene, may be produced. Thus, the invention includes each andevery possible variation of polynucleotide sequence that could be madeby selecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the polynucleotide sequence of naturally occurringBNO1, and all such variations are to be considered as being specificallydisclosed.

The polynucleotides of this invention include RNA, cDNA, genomic DNA,synthetic forms, and mixed polymers, both sense and antisense strands,and may be chemically or biochemically modified, or may containnon-natural or derivatised nucleotide bases as will be appreciated bythose skilled in the art. Such modifications include labels,methylation, intercalators, alkylators and modified linkages. In someinstances it may be advantageous to produce nucleotide sequencesencoding BNO1 or its derivatives possessing a substantially differentcodon usage than that of the naturally occurring BNO1. For example,codons may be selected to increase the rate of expression of the peptidein a particular prokaryotic or eukaryotic host corresponding with thefrequency that particular codons are utilized by the host. Other reasonsto alter the nucleotide sequence encoding BNO1 and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA molecules, which encodeBNO1 and its derivatives, or fragments thereof, entirely by syntheticchemistry. Synthetic sequences may be inserted into expression vectorsand cell systems that contain the necessary elements for transcriptionaland translational control of the inserted coding sequence in a suitablehost. These elements may include regulatory sequences, promoters, 5′ and3′ untranslated regions and specific initiation signals (such as an ATGinitiation codon and Kozak consensus sequence) which allow moreefficient translation of sequences encoding BNO1. In cases where thecomplete BNO1 coding sequence including its initiation codon andupstream regulatory sequences are inserted into the appropriateexpression vector, additional control signals may not be needed.However, in cases where only coding sequence, or a fragment thereof, isinserted, exogenous translational control signals as described aboveshould be provided by the vector. Such signals may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate for the particularhost cell system used (Scharf et al., 1994).

The present invention allows for the preparation of purified BNO1polypeptide or protein, from the polynucleotides of the presentinvention or variants thereof. In order to do this, host cells may betransfected with a DNA molecule as described above. Typically said hostcells are transfected with an expression vector comprising a DNAmolecule according to the invention. A variety of expression vector/hostsystems may be utilized to contain and express sequences encoding BNO1.These include, but are not limited to, microorganisms such as bacteriatransformed with plasmid or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); or mouse or otheranimal or human tissue cell systems. Mammalian cells can also be used toexpress the BNO1 protein using various expression vectors includingplasmid, cosmid and viral systems such as adenoviral, retroviral orvaccinia virus expression systems. The invention is not limited by thehost cell employed.

The polynucleotide sequences, or variants thereof, of the presentinvention can be stably expressed in cell lines to allow long termproduction of recombinant proteins in mammalian systems. Sequencesencoding BNO1 can be transformed into cell lines using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. The selectable marker confers resistance to a selectiveagent, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be propagated using tissue culturetechniques appropriate to the cell type.

The protein produced by a transformed cell may be secreted or retainedintracellularly depending on the sequence and/or the vector used. Aswill be understood by those of skill in the art, expression vectorscontaining polynucleotides which encode BNO1 may be designed to containsignal sequences which direct secretion of BNO1 through a prokaryotic oreukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, glycosylation,phosphorylation, and acylation. Post-translational cleavage of a“prepro” form of the protein may also be used to specify proteintargeting, folding, and/or activity. Different host cells havingspecific cellular machinery and characteristic mechanisms forpost-translational activities (e.g., CHO or HeLa cells), are availablefrom the American Type Culture Collection (ATCC) and may be chosen toensure the correct modification and processing of the foreign protein.

When large quantities of BNO1 are needed such as for antibodyproduction, vectors which direct high levels of expression of BNO1 maybe used such as those containing the T5 or T7 inducible bacteriophagepromoter. The present invention also includes the use of the expressionsystems described above in generating and isolating fusion proteinswhich contain important functional domains of the protein. These fusionproteins are used for binding, structural and functional studies as wellas for the generation of appropriate antibodies.

In order to express and purify the protein as a fusion protein, theappropriate BNO1 cDNA sequence is inserted into a vector which containsa nucleotide sequence encoding another peptide (for example,glutathionine succinyl transferase) . The fusion protein is expressedand recovered from prokaryotic or eukaryotic cells. The fusion proteincan then be purified by affinity chromatography based upon the fusionvector sequence and the BNO1 protein obtained by enzymatic cleavage ofthe fusion protein.

Fragments of BNO1 may also be produced by direct peptide synthesis usingsolid-phase techniques. Automated synthesis may be achieved by using theABI 431A Peptide Synthesizer (Perkin-Elmer). Various fragments of BNO1may be synthesized separately and then combined to produce thefull-length molecule.

According to the invention there is provided an isolated polypeptidecomprising the amino acid sequence set forth in SEQ ID Numbers: 2 or 4.

According to a still further aspect of the invention there is providedan isolated polypeptide, comprising the amino acid sequence set forth inSEQ ID Numbers: 2 or 4, or a fragment thereof, that forms part of aubiquitin-ligase complex involved in protein degradation throughubiquitination.

The invention also encompasses an isolated polypeptide that forms partof a ubiquitin-ligase complex involved in protein degradation throughubiquitination that has at least 70%, preferably 85%, and morepreferably 95%, identity with the amino acid sequence set forth in SEQID Numbers: 2 or 4.

Preferably, sequence identity is determined using the BLASTP algorithmwith the BLOSSUM62 default matrix.

Also envisaged is an isolated polypeptide consisting of the amino acidsequence set forth in SEQ ID Numbers: 2 or 4.

In a further aspect of the invention there is provided a method ofpreparing a polypeptide as described above, comprising the steps of:

(1) culturing the host cells under conditions effective for productionof the polypeptide; and

(2) harvesting the polypeptide.

Substantially purified BNO1 protein or fragments thereof can then beused in further biochemical analyses to establish secondary and tertiarystructure for example by x-ray crystallography of BNO1 protein or bynuclear magnetic resonance (NMR). Determination of structure allows forthe rational design of pharmaceuticals to interact with the protein,alter protein charge configuration or charge interaction with otherproteins, or to alter its function in the cell.

The BNO1 gene has been identified from a region of restricted LOH seenin breast and prostate cancer and appears to be down regulated in itsexpression in cancer cell lines derived from these tissues. In addition,chemical and structural similarity in the context of sequences andmotifs, exists between regions of BNO1 and F-box proteins. F-boxproteins are the substrate recognition components of one class ofubiquitin-E3 ligases, the so called “SCF” class, which are involved inthe degradation of proteins through ubiquitination and subsequentproteolysis carried out by the proteasome. To date, proteins shown to beregulated by this mechanism include oncogenes, tumour suppressor genes,transcription factors and other signalling molecules. These proteinsinfluence many cellular processes such as modulation of the immune andinflammatory responses, development and differentiation, as well asprocesses that are involved in cancer development such as cell-cycleregulation and apoptosis. BNO1 has also been shown to interact withSkp1, an essential component of SCF ubiquitin-E3 ligases.

A strong precedent for a tumour suppressor protein belonging to theubiquitin-proteasome degradation system has previously been provided bythe VHL gene. This gene has been demonstrated to associate with elonginC, elongin B, and cullin-2 in a complex termed VCB-CUL-2. Thismultiprotein complex exhibits structural and functional similarity toSCF ubiquitin ligases and has been shown to be involved in theubiquitination of VHL substrates.

Collectively, this information suggests BNO1 is involved in theprocesses that lead to cancer, particularly breast and prostatecarcinoma, most likely through its role in the ubiquitination ofproteins involved in important cellular functions such as cell cycleregulation. As BNO1 is expressed in many tissue types, alterations inBNO1 function may also cause pathologies in these tissues throughconsequential abnormalities in the ubiquitination process.

With the identification of the BNO1 nucleotide and protein sequence,probes and antibodies raised to the gene can be used in a variety ofhybridisation and immunological assays to screen for and detect thepresence of either a normal or mutated gene or gene product. In additionthe nucleotide and protein sequence of the BNO1 gene provided in thisinvention enables therapeutic methods for the treatment of all diseasesassociated with abnormalities of BNO1 function, including cancer,immune/inflammatory disease and neurological disorders, and also enablesmethods for the diagnosis or prognosis of all diseases associated withabnormalities of BNO1 function.

Examples of such disorders include, but are not limited to, cancers,immune/inflammatory disorders and neurological disorders. Cancersinclude adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the breast, prostate,liver, ovary, head and neck, heart, brain, pancreas, lung, skeletalmuscle, kidney, colon, uterus, testis, adrenal gland, blood, germ cells,placenta, synovial membrane, tonsil, cervix, lymph tissue, skin,bladder, spinal cord, thyroid gland and stomach. Other cancers mayinclude those of the bone, bone marrow, gall bladder, ganglia,gastrointestinal tract, parathyroid, penis, salivary glands, spleen andthymus. Immune/inflammatory disorders include acquired immunodeficiencysyndrome (AIDS), Addison's disease, adult respiratory distress syndrome,allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,atherosclerosis. autoimmune hemolytic anemia, autoimmune thyroiditis,autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy(APECED), bronchitis, cholecystitis, contact dermatitis, Crohn'sdisease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetesmellitus, emphysema, episodic lymphopenia with lymphocytotoxins,erythroblastosis fetalis, erythema nodosum, atrophic gastritis,glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,multiple sclerosis, myasthenia gravis, myocardial or pericardialinflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis,psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma,Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,systemic sclerosis, thrombocytopenic purpura, ulcerative colitis,uveitis, Werner syndrome, complications of wound healing (eg scarring),cancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma.Neurological disorders may include Parkinson's disease and Alzheimer'sdisease.

In the treatment of diseases associated with decreased BNO1 expressionand/or activity, it is desirable to increase the expression and/oractivity of BNO1. In the treatment of disorders associated withincreased BNO1 expression and/or activity, it is desirable to decreasethe expression and/or activity of BNO1.

Enhancing BNO1 Gene or Protein Function

Enhancing, stimulating or re-activating BNO1 gene or protein functioncan be achieved in a variety of ways. In one aspect of the inventionadministration of an isolated DNA molecule, as described above, to asubject in need of such treatment may be initiated.

Typically, BNO1 is administered to a subject to treat or prevent adisorder associated with decreased activity and/or expression of BNO1.

In a further aspect, there is provided the use of an isolated DNAmolecule, as described above, in the manufacture of a medicament for thetreatment of a disorder associated with decreased activity and/orexpression of BNO1.

Typically, a vector capable of expressing BNO1 or a fragment orderivative thereof may be administered to a subject to treat or preventa disorder associated with decreased activity and/or expression of TSG18including, but not limited to, those described above. Transducingretroviral vectors are often used for somatic cell gene therapy becauseof their high efficiency of infection and stable integration andexpression. The full length BNO1 gene, or portions thereof, can becloned into a retroviral vector and driven from its endogenous promoteror from the retroviral long terminal repeat or from a promoter specificfor the target cell type of interest. Other viral vectors can be usedand include, as is known in the art, adenoviruses, adeno-associatedvirus, vaccinia virus, papovaviruses, lentiviruses and retroviruses ofavian, murine and human origin.

Gene therapy would be carried out according to established methods(Friedman, 1991; Culver, 1996). A vector containing a copy of the BNO1gene linked to expression control elements and capable of replicatinginside the cells is prepared. Alternatively the vector may bereplication deficient and may require helper cells or helper virus forreplication and virus production and use in gene therapy.

Gene transfer using non-viral methods of infection can also be used.These methods include direct injection of DNA, uptake of naked DNA inthe presence of calcium phosphate, electroporation, protoplast fusion orliposome delivery. Gene transfer can also be achieved by delivery as apart of a human artificial chromosome or receptor-mediated genetransfer. This involves linking the DNA to a targeting molecule thatwill bind to specific cell-surface receptors to induce endocytosis andtransfer of the DNA into mammalian cells. One such technique usespoly-L-lysine to link asialoglycoprotein to DNA. An adenovirus is alsoadded to the complex to disrupt the lysosomes and thus allow the DNA toavoid degradation and move to the nucleus. Infusion of these particlesintravenously has resulted in gene transfer into hepatocytes.

In affected subjects that express a mutated form of BNO1 it may bepossible to prevent the disorder by introducing into the affected cellsa wild-type copy of the gene such that it recombines with the mutantgene. This requires a double recombination event for the correction ofthe gene mutation. Vectors for the introduction of genes in these waysare known in the art, and any suitable vector may be used.Alternatively, introducing another copy of the gene bearing a secondmutation in that gene may be employed so as to negate the original genemutation and block any negative effect.

In affected subjects that have decreased expression of BNO1, a mechanismof down-regulation may be abnormal methylation of the CpG island presentin the 5′ end of the gene. Therefore, in an alternative approach totherapy, administration of agents that remove BNO1 promoter methylationwill reactivate BNO1 gene expression and may suppress the associateddisease phenotype.

In a further aspect, a suitable agonist may also include a smallmolecule or peptide that can mimic the function of wild-type BNO1.

Inhibiting BNO1 Gene or Protein Function

Inhibiting the function of a mutated gene or protein can be achieved ina variety of ways. In one aspect of the invention there is provided amethod of treating a disorder associated with increased activity and/orexpression of BNO1, comprising administering an antagonist of BNO1 to asubject in need of such treatment.

In still another aspect of the invention there is provided the use of anantagonist of BNO1 in the manufacture of a medicament for the treatmentof a disorder associated with increased activity and/or expression ofBNO1.

Such disorders may include, but are not limited to, those discussedabove. In one aspect of the invention an isolated DNA molecule, which isthe complement of any one of the DNA molecules described above and whichencodes an RNA molecule that hybridises with the mRNA encoded by BNO1,may be administered to a subject in need of such treatment.

In a still further aspect of the invention there is provided the use ofan isolated DNA molecule which is the complement of a DNA molecule ofthe invention and which encodes an RNA molecule that hybridises with themRNA encoded by BNO1, in the manufacture of a medicament for thetreatment of a disorder associated with increased activity and/orexpression of BNO1.

Typically, a vector expressing the complement of the polynucleotideencoding BNO1 may be administered to a subject to treat or prevent adisorder associated with increased activity and/or expression of BNO1including, but not limited to, those described above. Antisensestrategies may use a variety of approaches including the use ofantisense oligonucleotides, ribozymes, DNAzymes, injection of antisenseRNA and transfection of antisense RNA expression vectors. Many methodsfor introducing vectors into cells or tissues are available and equallysuitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy,vectors may be introduced into stem cells taken from the patient andclonally propagated for autologous transplant back into that samepatient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (For example, see Goldman et al., 1997).

According to still another aspect of the invention, there is provided amethod of treating a disorder associated with increased activity and/orexpression of BNO1 comprising administering an antagonist of BNO1 to asubject in need of such treatment.

In still another aspect of the invention there is provided the use of anantagonist of BNO1 in the manufacture of a medicament for the treatmentof a disorder associated with increased activity and/or expression ofBNO1.

Such disorders may include, but are not limited to, those discussedabove. In one aspect purified protein according to the invention may beused to produce antibodies which specifically bind BNO1. Theseantibodies may be used directly as an antagonist or indirectly as atargeting or delivery mechanism for bringing a pharmaceutical agent tocells or tissues that express BNO1. Such antibodies may include, but arenot limited to, polyclonal, monoclonal, chimeric and single chainantibodies as would be understood by the person skilled in the art.

For the production of antibodies, various hosts including rabbits, rats,goats, mice, humans, and others may be immunized by injection with aprotein of the invention or with any fragment or oligopeptide thereof,which has immunogenic properties. Various adjuvants may be used toincrease immunological response and include, but are not limited to,Freund's, mineral gels such as aluminum hydroxide, and surface-activesubstances such as lysolecithin. Adjuvants used in humans include BCG(bacilli Calmette-Guerin) and Corynebacterium parvum.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to BNO1 have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of amino acids fromthese proteins may be fused with those of another protein, such as KLH,and antibodies to the chimeric molecule may be produced.

Monoclonal antibodies to BNO1 may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (For example, see Kohler et al., 1975; Kozbor et al., 1985;Cote et al., 1983; Cole et al., 1984).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature. (Forexample, see Orlandi et al., 1989; Winter et al., 1991).

Antibody fragments which contain specific binding sites for BNO1 mayalso be generated. For example, such fragments include, F(ab′)2fragments produced by pepsin digestion of the antibody molecule and Fabfragments generated by reducing the disulfide bridges of the F(ab′)2fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity. (For example, see Huse et al., 1989).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between a protein and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes is preferred, but a competitive bindingassay may also be employed.

In a still further aspect the invention provides a method for thetreatment of a disorder shown to be associated with abnormal activityand/or expression of BNO1, comprising administering a nucleic acidmolecule, antibody or compound as described above, to a subject in needof such treatment.

In another aspect the invention provides the use of a nucleic acidmolecule, antibody or compound as described above, in the manufacture ofa medicament for the treatment of a disorder shown to be associated withabnormal activity and/or expression of BNO1.

In a further aspect a pharmaceutical composition comprising a nucleicacid molecule, antibody or compound as described above, and apharmaceutically acceptable carrier may be administered.

The pharmaceutical composition may be administered to a subject to treator prevent a disorder associated with abnormal activity and/orexpression of BNO1 including, but not limited to, those provided above.Pharmaceutical compositions in accordance with the present invention areprepared by mixing BNO1 or active fragments or variants thereof havingthe desired degree of purity, with acceptable carriers, excipients, orstabilizers which are well known. Acceptable carriers, excipients orstabilizers are nontoxic at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including absorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitrol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

In further embodiments, any of the genes, peptides, antagonists,antibodies, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents may be made by those skilled in theart, according to conventional pharmaceutical principles. Thecombination of therapeutic agents may act synergistically to effect thetreatment or prevention of the various disorders described above. Usingthis approach, therapeutic efficacy with lower dosages of each agent maybe possible, thus reducing the potential for adverse side effects.

Drug Screening

According to still another aspect of the invention, peptides of theinvention, particularly purified BNO1 polypeptides, and cells expressingthese are useful for screening of candidate pharmaceutical agents in avariety of techniques for the treatment of disorders associated withBNO1 dysfunction. Such techniques include, but are not limited to,utilising eukaryotic or prokaryotic host cells that are stablytransformed with recombinant molecules expressing the BNO1 polypeptideor fragment thereof, preferably in competitive binding assays. Bindingassays will measure for the formation of complexes between the BNO1polypeptide, or fragments thereof, and the agent being tested, or willmeasure the degree to which an agent being tested will interfere withthe formation of a complex between the BNO1 polypeptide, or fragmentthereof, and a known ligand, particularly other members of the SCFcomplex and BNO1 substrates targeted for ubiquitination.

Another technique for drug screening provides high-throughput screeningfor compounds having suitable binding affinity to the BNO1 polypeptide(see PCT published application WO84/03564). In this stated technique,large numbers of small peptide test compounds can be synthesised on asolid substrate and can be assayed through BNO1 polypeptide binding andwashing. Bound BNO1 polypeptide is then detected by methods well knownin the art. In a variation of this technique, purified polypeptides canbe coated directly onto plates to identify interacting test compounds.

An additional method for drug screening involves the use of hosteukaryotic cell lines which carry mutations in the BNO1 gene. The hostcell lines are also defective at the polypeptide level. Other cell linesmay be used where the gene expression of BNO1 can be switched off. Thehost cell lines or cells are grown in the presence of various drugcompounds and the rate of growth of the host cells is measured todetermine if the compound is capable of regulating the growth ofdefective cells.

BNO1 polypeptide may also be used for screening compounds developed as aresult of combinatorial library technology. This provides a way to testa large number of different substances for their ability to modulateactivity of a polypeptide. The use of peptide libraries is preferred(see patent WO97/02048) with such libraries and their use known in theart.

A substance identified as a modulator of polypeptide function may bepeptide or non-peptide in nature. Non-peptide “small molecules” areoften preferred for many in vivo pharmaceutical applications. Inaddition, a mimic or mimetic of the substance may be designed forpharmaceutical use. The design of mimetics based on a knownpharmaceutically active compound (“lead” compound) is a common approachto the development of novel pharmaceuticals. This is often desirablewhere the original active compound is difficult or expensive tosynthesise or where it provides an unsuitable method of administration.In the design of a mimetic, particular parts of the original activecompound that are important in determining the target property areidentified. These parts or residues constituting the active region ofthe compound are known as its pharmacophore. Once found, thepharmacophore structure is modelled according to its physical propertiesusing data from a range of sources including x-ray diffraction data andNMR. A template molecule is then selected onto which chemical groupswhich mimic the pharmacophore can be added. The selection can be madesuch that the mimetic is easy to synthesise, is likely to bepharmacologically acceptable, does not degrade in vivo and retains thebiological activity of the lead compound. Further optimisation ormodification can be carried out to select one or more final mimeticsuseful for in vivo or clinical testing.

It is also possible to isolate a target-specific antibody and then solveits crystal structure. In principle, this approach yields apharmacophore upon which subsequent drug design can be based asdescribed above. It may be possible to avoid protein crystallographyaltogether by generating anti-idiotypic antibodies (anti-ids) to afunctional, pharmacologically active antibody. As a mirror image of amirror image, the binding site of the anti-ids would be expected to bean analogue of the original binding site. The anti-id could then be usedto isolate peptides from chemically or biologically produced peptidebanks.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

Diagnostic and Prognostic Applications

Polynucleotide sequences encoding BNO1 may be used for the diagnosis orprognosis of disorders associated with BNO1 dysfunction, or apredisposition to such disorders. Examples of such disorders include,but are not limited to, cancers, immune/inflammatory disorders andneurological disorders. Cancers include adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, inparticular, cancers of the breast, prostate, liver, ovary, head andneck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon,uterus, testis, adrenal gland, blood, germ cells, placenta, synovialmembrane, tonsil, cervix, lymph tissue, skin, bladder, spinal cord,thyroid gland and stomach. Other cancers may include those of the bone,bone marrow, gall bladder, ganglia, gastrointestinal tract, parathyroid,penis, salivary glands, spleen and thymus. Immune/inflammatory disordersinclude acquired immunodeficiency syndrome (AIDS), Addison's disease,adult respiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis. autoimmune hemolyticanemia, autoimmune thyroiditis, autoimmunepolyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cysticfibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosisfetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of wound healing (eg scarring), cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, and trauma.Neurological disorders may include Parkinson's disease and Alzheimer'sdisease.

Diagnosis or prognosis may be used to determine the severity, type orstage of the disease state in order to initiate an appropriatetherapeutic intervention.

In another embodiment of the invention, the polynucleotides that may beused for diagnostic or prognostic purposes include oligonucleotidesequences, genomic DNA and complementary RNA and DNA molecules. Thepolynucleotides may be used to detect and quantitate gene expression inbiopsied tissues in which mutations in BNO1 or abnormal expression ofBNO1 may be correlated with disease. Genomic DNA used for the diagnosisor prognosis may be obtained from body cells, such as those present inthe blood, tissue biopsy, surgical specimen, or autopsy material. TheDNA may be isolated and used directly for detection of a specificsequence or may be amplified by the polymerase chain reaction (PCR)prior to analysis. Similarly, RNA or cDNA may also be used, with orwithout PCR amplification. To detect a specific nucleic acid sequence,direct nucleotide sequencing, reverse transcriptase PCR (RT-PCR),hybridization using specific oligonucleotides, restriction enzyme digestand mapping, PCR mapping, RNAse protection, and various other methodsmay be employed. Oligonucleotides specific to particular sequences canbe chemically synthesized and labelled radioactively ornon-radioactively and hybridised to individual samples immobilized onmembranes or other solid-supports or in solution. The presence, absenceor excess expression of BNO1 may then be visualized using methods suchas autoradiography, fluorometry, or colorimetry.

In a particular aspect, the nucleotide sequences encoding BNO1 may beuseful in assays that detect the presence of associated disorders,particularly those mentioned previously. The nucleotide sequencesencoding BNO1 may be labelled by standard methods and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the patient sample issignificantly altered in comparison to a control sample then thepresence of altered levels of nucleotide sequences encoding BNO1 in thesample indicates the presence of the associated disorder. Such assaysmay also be used to evaluate the efficacy of a particular therapeutictreatment regimen in animal studies, in clinical trials, or to monitorthe treatment of an individual patient.

In order to provide a basis for the diagnosis or prognosis of a disordershown to be associated with a mutation in BNO1, the nucleotide sequenceof the BNO1 gene can be compared between normal tissue and diseasedtissue in order to establish whether the patient expresses a mutantgene.

In order to provide a basis for the diagnosis or prognosis of a disordershown to be associated with abnormal expression of BNO1, a normal orstandard profile for expression is established. This may be accomplishedby combining body fluids or cell extracts taken from normal subjects,either animal or human, with a sequence, or a fragment thereof, encodingBNO1, under conditions suitable for hybridization or amplification.Standard hybridization may be quantified by comparing the valuesobtained from normal subjects with values from an experiment in which aknown amount of a substantially purified polynucleotide is used. Anothermethod to identify a normal or standard profile for expression of BNO1is through quantitative RT-PCR studies. RNA isolated from body cells ofa normal individual, particularly RNA isolated from tumour cells, isreverse transcribed and real-time PCR using oligonucleotides specificfor the BNO1 gene is conducted to establish a normal level of expressionof the gene.

Standard values obtained in both these examples may be compared withvalues obtained from samples from patients who are symptomatic for adisorder. Deviation from standard values is used to establish thepresence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays or quantitative RT-PCR studies may berepeated on a regular basis to determine if the level of expression inthe patient begins to approximate that which is observed in the normalsubject. The results obtained from successive assays may be used to showthe efficacy of treatment over a period ranging from several days tomonths.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding BNO1 or closely related molecules may be used to identifynucleic acid sequences which encode BNO1. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification willdetermine whether the probe identifies only naturally occurringsequences encoding BNO1, allelic variants, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably have at least 50% sequence identity to any of the BNO1encoding sequences. The hybridization probes of the subject inventionmay be DNA or RNA and may be derived from the sequence of SEQ IDNumbers: 1 or 3 or from genomic sequences including promoters,enhancers, and introns of the BNO1 gene (SEQ ID Numbers: 5-11).

Means for producing specific hybridization probes for DNAs encoding BNO1include the cloning of polynucleotide sequences encoding BNO1 or BNO1derivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, and are commercially available. Hybridizationprobes may be labelled by radionuclides such as ³²P or 35S, or byenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, or other methods known in the art.

According to a further aspect of the invention there is provided the useof a polypeptide as described above in the diagnosis or prognosis of adisorder shown to be associated with BNO1, or a predisposition to suchdisorders.

When a diagnostic or prognostic assay is to be based upon the BNO1protein, a variety of approaches are possible. For example, diagnosis orprognosis can be achieved by monitoring differences in theelectrophoretic mobility of normal and mutant proteins. Such an approachwill be particularly useful in identifying mutants in which chargesubstitutions are present, or in which insertions, deletions orsubstitutions have resulted in a significant change in theelectrophoretic migration of the resultant protein. Alternatively,diagnosis may be based upon differences in the proteolytic cleavagepatterns of normal and mutant proteins, differences in molar ratios ofthe various amino acid residues, or by functional assays demonstratingaltered function of the gene products.

In another aspect, antibodies that specifically bind BNO1 may be usedfor the diagnosis or prognosis of disorders characterized by abnormalexpression of BNO1, or in assays to monitor patients being treated withBNO1 or agonists, antagonists, or inhibitors of BNO1. Antibodies usefulfor diagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic or prognostic assays for BNO1 includemethods that utilize the antibody and a label to detect BNO1 in humanbody fluids or in extracts of cells or tissues. The antibodies may beused with or without modification, and may be labelled by covalent ornon-covalent attachment of a reporter molecule.

A variety of protocols for measuring BNO1, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of BNO1 expression. Normal or standard values for BNO1expression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toBNO1 under conditions suitable for complex formation. The amount ofstandard complex formation may be quantitated by various methods,preferably by photometric means. Quantities of BNO1 expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

Once an individual has been diagnosed with a disorder, effectivetreatments can be initiated. These may include administering a selectiveagonist to the mutant BNO1 so as to restore its function to a normallevel or introduction of wild-type BNO1, particularly through genetherapy approaches as described above. Typically, a vector capable ofexpressing the appropriate full-length BNO1 gene or a fragment orderivative thereof may be administered. In an alternative approach totherapy, substantially purified BNO1 polypeptide and a pharmaceuticallyacceptable carrier may be administered as described above or drugs whichcan replace the function of, or mimic the action of BNO1 may beadministered.

In the treatment of disorders shown to be associated with increased BNO1expression and/or activity, the affected individual may be treated witha selective antagonist such as an antibody to the relevant protein or anantisense (complement) probe to the corresponding gene as describedabove, or through the use of drugs which may block the action of BNO1.

Microarray

In further embodiments, complete cDNAs, oligonucleotides or longerfragments derived from any of the polynucleotide sequences describedherein may be used as targets in a microarray. The microarray can beused to monitor the expression level of large numbers of genessimultaneously and to identify genetic variants, mutations, andpolymorphisms. This information may be used to determine gene function,to understand the genetic basis of a disorder, to diagnose or prognose adisorder, and to develop and monitor the activities of therapeuticagents. Microarrays may be prepared, used, and analyzed using methodsknown in the art. (For example, see Schena et al., 1996; Heller et al.,1997).

Transformed Hosts

The present invention also provides for the production of geneticallymodified (knock-out, knock-in and transgenic) , non-human animal modelstransformed with the DNA molecules of the invention. These animals areuseful for the study of the BNO1 gene function, to study the mechanismsof disease as related to the BNO1 gene, for the screening of candidatepharmaceutical compounds, for the creation of explanted mammalian cellcultures which express the protein or mutant protein and for theevaluation of potential therapeutic interventions.

The BNO1 gene may have been inactivated by knock-out deletion, andknock-out genetically modified non-human animals are therefore provided.

Animal species which are suitable for use in the animal models of thepresent invention include, but are not limited to, rats, mice, hamsters,guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, and non-humanprimates such as monkeys and chimpanzees. For initial studies,genetically modified mice and rats are highly desirable due to theirrelative ease of maintenance and shorter life spans. For certainstudies, transgenic yeast or invertebrates may be suitable and preferredbecause they allow for rapid screening and provide for much easierhandling. For longer term studies, non-human primates may be desired dueto their similarity with humans.

To create an animal model for mutated BNO1 several methods can beemployed. These include generation of a specific mutation in ahomologous animal gene, insertion of a wild type human gene and/or ahumanized animal gene by homologous recombination, insertion of a mutant(single or multiple) human gene as genomic or minigene cDNA constructsusing wild type or mutant or artificial promoter elements or insertionof artificially modified fragments of the endogenous gene by homologousrecombination. The modifications include insertion of mutant stopcodons, the deletion of DNA sequences, or the inclusion of recombinationelements (lox p sites) recognized by enzymes such as Cre recombinase.

To create a transgenic mouse, which is preferred, a mutant version ofBNO1 can be inserted into a mouse germ line using standard techniques ofoocyte microinjection or transfection or microinjection into embryonicstem cells. Alternatively, if it is desired to inactivate or replace theendogenous BNO1 gene, homologous recombination using embryonic stemcells may be applied.

For oocyte injection, one or more copies of the mutant or wild type BNO1gene can be inserted into the pronucleus of a just-fertilized mouseoocyte. This oocyte is then reimplanted into a pseudo-pregnant fostermother. The liveborn mice can then be screened for integrants usinganalysis of tail DNA for the presence of human BNO1 gene sequences. Thetransgene can be either a complete genomic sequence injected as a YAC,BAC, PAC or other chromosome DNA fragment, a cDNA with either thenatural promoter or a heterologous promoter, or a minigene containingall of the coding region and other elements found to be necessary foroptimum expression.

According to still another aspect of the invention there is provided theuse of genetically modified non-human animals as described above for thescreening of candidate pharmaceutical compounds.

The identification of the nucleotide and amino acid sequence of bothisoforms of BNO1 enables the identification of BNO1-specific proteinsubstrates using protein interaction studies such as the yeasttwo-hybrid analysis as would be understood by those skilled in the art.These protein substrates would be targets for degradation viaubiquitination mediated by the BNO1-containing ubiquitin-E3 ligase. Eachisoform of BNO1 may share common protein substrates or may interact withisoform-specific substrates.

In one aspect of the invention there is provided a complex of wild-typeBNO1 and a BNO1-specific substrate that is targeted for degradation byubiquitination.

In a still further aspect of the invention there is provided a complexof BNO1 and proteins of the ubiquitin-E3 ligase complex.

According to a still further aspect of the invention there is provided acomplex of wild-type BNO1 and the Skp1 protein.

In a still further aspect of the invention there is provided a nucleicacid encoding a mutant BNO1 polypeptide which cannot form a complex withwild-type proteins with which wild-type BNO1 does form a complex.Typically one of these proteins is Skp1 while others are BNO1-specificprotein substrates targeted for degradation by ubiquitination.

According to a still further aspect of the invention there is provided amutant BNO1 polypeptide which cannot form a complex with wild-typeproteins with which wild-type BNO1 does form a complex. Typically one ofthese proteins is Skp1 while others are BNO1-specific protein substratestargeted for degradation by ubiquitination.

In a still further aspect of the present invention there is provided theuse of complexes as described above in screening for candidatepharmaceutical compounds. One may also screen for a drug which replacesthe activity of BNO1 in a patient deficient in BNO1.

It will be clearly understood that, although a number of prior artpublications are referred to herein, this reference does not constitutean admission that any of these documents forms part of the commongeneral knowledge in the art, in Australia or in any other country.Throughout this specification and the claims, the words “comprise”,“comprises” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of tumours with interstitial andterminal allelic loss on chromosome arm 16q in the two series of tumoursamples. Polymorphic markers are listed according to their order on 16qfrom centromere to telomere and the markers used for each series areindicated by X. Tumour identification numbers are shown at the top ofeach column. At the right of the figure, the three smallest regions ofloss of heterozygosity are indicated.

FIG. 2. Northern blot analysis of the BNO1 gene. The size of the BNO1gene in kilobases is indicated by an arrow on the left of the Northern.The blot contained RNA from the following tissues: 1: Mammary gland; 2:Bone marrow; 3: Testis; 4: Ovary; 5: Uterus; 6: Prostate; 7: Stomach; 8:Bladder; 9: Spinal cord; 10: Brain; 11: Pancreas; 12: Thyroid. A singleband of approximately 3.6 Kb was seen in all tissues except bone marrow.Strongest expression of the gene was seen in the brain.

FIG. 3. BNO1 F-box sequence alignment compared with the F-box consensussequence as reported by Kipreos and Pagano, (2000). The single letteramino-acid code is used. Bold capital letters indicate residues found inover 40% of F-box sequences; non-bold capital letters indicate residuesfound in 20-40% of F-box sequences; bold, lower case letters indicateresidues found in 15-19% of the F-boxes; non-bold lower case lettersindicate residues found in 10-14% of F-boxes. The top line representsthe F-box motif of BNO1 indicating a high degree of homology with theconsensus.

FIG. 4. Quantitative RT-PCR expression analysis of the BNO1 gene inbreast cancer cell lines. BNO1 copy numbers in normalized normal mammarygland (breast) cDNA were arbitrarily set to a ‘baseline’ of 1.Oe+06(empty bar) . Breast cancer cell lines and other normal tissue cDNA copynumbers were calculated relative to the ‘baseline’. Grey filled barsrepresent amplicon fold expression down-regulation compared to thebaseline reference, while black filled bars represent amplicon foldexpression up-regulation from the baseline reference. Note: replicatecell lines (a and b) represent independent cell cultures, total RNAisolation and reverse transcription reactions. Replicates served asanother level of control to monitor the variability in gene expressionresulting from differences in cell confluency, total RNA integrity andreverse transcription efficiencies.

MODES FOR PERFORMING THE INVENTION EXAMPLE 1 Collection of Breast CancerPatient Material

Two series of breast cancer patients were analysed for this study.Histopathological classification of each tumour specimen was carried outby our collaborators according to World Health Organisation criteria(WHO, 1981). Patients were graded histopathologically according to themodified Bloom and Richardson method (Elston and Ellis, 1990) andpatient material was obtained upon approval of local Medical EthicsCommittees. Tumour tissue DNA and peripheral blood DNA from the sameindividual was isolated as previously described (Devilee et al., 1991)using standard laboratory protocols.

Series 1 consisted of 189 patients operated on between 1986 and 1993 inthree Dutch hospitals, a Dutch University and two peripheral centres.Tumour tissue was snap frozen within a few hours of resection. For DNAisolation, a tissue block was selected only if it contained at least 50%of tumour cells following examination of haematoxilin and eosin stainedtissue sections by a pathologist. Tissue blocks that contained fewerthan 50% of tumour cells were omitted from further analysis.

Series 2 consisted of 123 patients operated on between 1987 and 1997 atthe Flinders Medical Centre in Adelaide, Australia. Of these, 87 werecollected as fresh specimens within a few hours of surgical resection,confirmed as malignant tissue by pathological analysis, snap frozen inliquid nitrogen, and stored at −70° C. The remaining 36 tumour tissuesamples were obtained from archival paraffin embedded tumour blocks.Prior to DNA isolation, tumour cells were microdissected from tissuesections mounted on glass slides so as to yield at least 80% tumourcells. In some instances, no peripheral blood was available such thatpathologically identified paraffin embedded non-malignant lymph nodetissue was used instead.

EXAMPLE 2 LOH Analysis of Chromosome 16q Markers in Breast CancerSamples

In order to identify the location of genes associated with breastcancer, LOH analysis of tumour samples was conducted. A total of 45genetic markers mapping to chromosome 16 were used for the LOH analysisof the breast tumour and matched normal DNA samples collected for thisstudy. FIG. 1 indicates for which tumour series they were used and theircytogenetic location. Details regarding all markers can be obtained fromthe Genome Database (GDB) at http://www.gdb.org. The physical order ofmarkers with respect to each other was determined from a combination ofinformation in GDB, by mapping on a chromosome 16 somatic cell hybridmap (Callen et al., 1995) and by genomic sequence information.

Four alternative methods were used for the LOH analysis:

1) For RFLP and VNTR markers, Southern blotting was used to test forallelic imbalance. These markers were used on only a subset of samples.Methods used were as previously described (Devilee et al., 1991). 2)Microsatellite markers were amplified from tumour and normal DNA usingthe polymerase chain reaction (PCR) incorporating standard methodologies(Weber and May, 1989; Sambrook et al., 1989). A typical reactionconsisted of 12 μl and contained 100 ng of template, 5 pmol of bothprimers, 0.2 mM of each dNTP, 1 μCurie [α-³²P]dCTP, 1.5 mM MgCl₂, 1.2 μlSupertaq buffer and 0.06 units of Supertaq (HT biotechnologies). APhosphor Imager type 445 SI (Molecular Dynamics, Sunnyvale, Calif.) wasused to quantify ambiguous results. In these cases, the AllelicImbalance Factor (AIF) was determined as the quotient of the peak heightratios from the normal and tumour DNA pair. The threshold for allelicimbalance was defined as a 40% reduction of one allele, agreeing with anAIF of ≧1.7 or ≦0.59. This threshold is in accordance with the selectionof tumour tissue blocks containing at least 50% tumour cells with a 10%error-range. The threshold for retention has been previously determinedto range from 0.76 to 1.3 (Devilee et al., 1994). This leaves a range ofAIFs (0.58-0.75 and 1.31-1.69) for which no definite decision has beenmade. This “grey area” is indicated by grey boxes in FIG. 1 and tumourswith only “grey area” values were discarded completely from theanalysis.

3) The third method for determining allelic imbalance was similar to thesecond method above, however radioactively labelled dCTP was omitted.Instead, PCR of polymorphic microsatellite markers was done with one ofthe PCR primers labelled fluorescently with FAM, TET or HEX. Analysis ofPCR products generated was on an ABI 377 automatic sequencer (PEBiosystems) using 6% polyacrylamide gels containing 8M urea. Peak heightvalues and peak sizes were analysed with the GeneScan programme (PEBiosystems). The same thresholds for allelic imbalance, retention andgrey areas were used as for the radioactive analysis.

4) An alternative fluorescent based system was also used. In thisinstance PCR primers were labelled with fluorescein orhexachlorofluorescein. PCR reaction volumes were 20 μl and included 100ng of template, 100 ng of each primer, 0.2 mM of each dNTP, 1-2 mMMgCl₂, 1×AmpliTaq Gold buffer and 0.8 units AmpliTaq Gold enzyme (PerkinElmer). Cycling conditions were 10 cycles of 94° C. for 30 seconds, 60°C. for 30 seconds, 72° C. for 1 minute, followed by 25 cycles of 94° C.30 seconds, 55° C. for 30 seconds, 72° C. for 1 minute, with a finalextension of 72° C. for 10 minutes. PCR amplimers were analysed on anABI 373 automated sequencer (PE Biosystems) using the GeneScan programme(PE Biosystems). The threshold range of AIF for allele retention wasdefined as 0.61-1.69, allelic loss as ≦0.5 or ≧2.0, or the “grey area”as 051-0.6 or 1.7-1.99.

The first three methods were applied to the first tumour series whilethe last method was adopted for the second series of tumour samples. Forstatistical analysis, a comparison of allelic imbalance data forvalidation of the different detection methods and of the differenttumour series was done using the Chi-square test.

The identification of the smallest region of overlap (SRO) involved inLOH is instrumental for narrowing down the location of the gene targetedby LOH. FIG. 1 shows the LOH results for tumour samples, which displayedsmall regions of loss (ie interstitial and telomeric LOH) and does notinclude samples that showed complex LOH (alternating loss and retentionof markers). When comparing the two sample sets at least threeconsistent regions emerge with two being at the telomere in band 16q24.3and one at 16q22.1. The region at 16q22.1 is defined by the markersD16S398 and D16S301 and is based on the interstitial LOH events seen inthree tumours from series 1 (239/335/478) and one tumour from series 2(237). At the telomere (16q24.2-16q24.3), the first region is defined bythe markers D16S498 and D16S3407 and is based on four tumours fromseries 2 (443/75/631/408) while the second region (16q24.3) extends fromD16S3407 to the telomere and is based on one tumour from series 1 (559)and three from series 2 (97/240/466). LOH limited to the telomere butinvolving both of the regions identified at this site could be found inan additional 17 tumour samples.

Other studies have shown that the long arm of chromosome 16 is also atarget for LOH in prostate, lung, hepatocellular, ovarian,rhabdomyosarcoma and Wilms' tumours. Detailed analysis of prostatecarcinomas has revealed an overlap in the smallest regions of LOH seenin this cancer to that seen with breast cancer which suggests that 16qharbours a gene implicated in many tumour types.

EXAMPLE 3 Construction of a Physical Map of 16q24.3

To identify novel candidate breast cancer genes mapping to the smallestregions of overlap at 16q24.3, a clone based physical map contigcovering this region was needed. At the start of this phase of theproject the most commonly used and readily accessible cloned genomic DNAfragments were contained in lambda, cosmid or YAC vectors. During theconstruction of whole chromosome 16 physical maps, clones from a numberof YAC libraries were incorporated into the map (Doggett et al., 1995).These included clones from a flow-sorted chromosome 16-specific YAClibrary (McCormick et al., 1993), from the CEPH Mark I and MegaYAClibraries and from a half-telomere YAC library (Riethman et al., 1989).Detailed STS and Southern analysis of YAC clones mapping at 16q24.3established that very few were localised between the CY2/CY3 somaticcell hybrid breakpoint and the long arm telomere. However, those thatwere located in this region gave inconsistent mapping results and weresuspected to be rearranged or deleted. Coupled with the fact that YACclones make poor sequencing substrates, and the difficulty in isolatingthe cloned human DNA, a physical map based on cosmid clones was theinitial preferred option.

A flow-sorted chromosome 16 specific cosmid library had previously beenconstructed (Longmire et al., 1993), with individual cosmid clonesgridded in high-density arrays onto nylon membranes. These filterscollectively contained ˜15,000 clones representing an approximately 5.5fold coverage of chromosome 16. Individual cosmids mapping to thecritical regions at 16q24.3 were identified by the hybridisation ofthese membranes with markers identified by this and previous studies tomap to the region. The strategy to align overlapping cosmid clones wasbased on their STS content and restriction endonuclease digestionpattern. Those clones extending furthest within each initial contig werethen used to walk along the chromosome by the hybridisation of the endsof these cosmids back to the high-density cosmid grids. This processcontinued until all initial contigs were linked and therefore the regiondefining the location of the breast cancer tumour suppressor genes wouldbe contained within the map. Individual cosmid clones representing aminimum tiling path in the contig were then used for the identificationof transcribed sequences by exon trapping, and for genomic sequencing.

Chromosome 16 was sorted from the mouse/human somatic cell hybrid CY18,which contains this chromosome as the only human DNA, and Sau3Apartially digested CY18 DNA was ligated into the BamHI cloning site ofthe cosmid sCOS-1 vector. All grids were hybridised and washed usingmethods described in Longmire et al. (1993). Briefly, the 10 filterswere pre-hybridised in 2 large bottles for at least 2 hours in 20 ml ofa solution containing 6×SSC; 10 mM EDTA (pH8.0); 10×Denhardt's; 1% SDSand 100 μg/ml denatured fragmented salmon sperm DNA at 65° C. Overnighthybridisations with [α-³²P]dCTP labelled probes were performed in 20 mlof fresh hybridisation solution at 65° C. Filters were washedsequentially in solutions of 2×SSC; 0.1% SDS (rinse at roomtemperature), 2×SSC; 0.1% SDS (room temperature for 15 minutes),0.1×SSC; 0.1% SDS (room temperature for 15 minutes), and b 0.1×SSC; 0.1%SDS (twice for 30 minutes at 50° C. if needed). Membranes were exposedat −70° C. for between 1 to 7 days.

Initial markers used for cosmid grid screening were those known to belocated below the somatic cell hybrid breakpoints CY2/CY3 and the longarm telomere (Callen et al., 1995). These included three genes, CMAR,DPEP1, and MC1R; the microsatellite marker D16S303; an end fragment fromthe cosmid 317E5, which contains the BBC1 gene; and four cDNA clones,yc81e09, yh09a04, D16S532E, and ScDNA-C113. The IMAGE consortium cDNAclone, yc81e09, was obtained through screening an arrayed normalisedinfant brain oligo-dT primed cDNA library (Soares et al., 1994), withthe insert from cDNA clone ScDNA-A55. Both the ScDNA-A55 and ScDNA-C113clones were originally isolated from a hexamer primed heteronuclear cDNAlibrary constructed from the mouse/human somatic cell hybrid CY18(Whitmore et al., 1994). The IMAGE cDNA clone yh09a04 was identifiedfrom direct cDNA selection of the cosmid 37B2 which was previously shownto map between the CY18A(D2) breakpoint and the 16q telomere. The EST,D16S532E, was also mapped to the same region. Subsequent to theseinitial screenings, restriction fragments representing the ends ofcosmids were used to identify additional overlapping clones.

Contig assembly was based on methods previously described (Whitmore etal., 1998). Later during the physical map construction, genomiclibraries cloned into BAC or PAC vectors (Genome Systems or RosewellPark Cancer Institute) became available. These libraries were screenedto aid in chromosome walking or when gaps that could not be bridged byusing the cosmid filters were encountered. All BAC and PAC filters werehybridised and washed according to manufacturers recommendations.Initially, membranes were individually pre-hybridised in large glassbottles for at least 2 hours in 20 ml of 6×SSC; 0.5% SDS; 5×Denhardt's;100 μg/ml denatured salmon sperm DNA at 65° C. Overnight hybridisationswith [α-³²P]dCTP labelled probes were performed at 65° C. in 20 ml of asolution containing 6×SSC; 0.5% SDS; 100 μg/ml denatured salmon spermDNA. Filters were washed sequentially in solutions of 2×SSC; 0.5% SDS(room temperature 5 minutes), 2×SSC; 0.1% SDS (room temperature 15minutes) and 0.1×SSC; 0.5% SDS (37° C. 1 hour if needed). PAC or BACclones identified were aligned to the existing contig based on theirrestriction enzyme pattern or formed unique contigs which were extendedby additional filter screens.

As the microsatellite D16S303 was known to be the most telomeric markerin the 16q24.3 region (Callen et al., 1995), fluorescence in situhybridisation (FISH) to normal metaphase chromosomes using whole cosmidsmapping in the vicinity of this marker, was used to define the telomericlimit for the contig. Whole cosmid DNA was nick translated withbiotin-14-dATP and hybridised in situ at a final concentration of 20ng/μl to metaphases from 2 normal males. The FISH method had beenmodified from that previously described (Callen et al., 1990).Chromosomes were stained before analysis with both propidium iodide (ascounter-stain) and DAPI (for chromosome identification). Images ofmetaphase preparations were captured by a cooled CCD camera using theCytoVision Ultra image collection and enhancement system (AppliedImaging Int. Ltd.). The cosmid 369E1 showed clear fluorescent signals atthe telomere of the long arm of chromosome 16. However, this probe alsogave clear signal at the telomeres of chromosomal arms 3q, 7p, 9q, 11p,and 17p. Conversely, the cosmid 439G8, which mapped proximal to D16S303,gave fluorescent signals only at 16qter with no consistent signaldetected at other telomeres. These results enabled us to establish themicrosatellite marker D16S303 as the boundary of the transition fromeuchromatin to the subtelomeric repeats, providing a telomeric limit tothe contig (Whitmore et al., 1998).

A high-density physical map consisting of cosmid, BAC and PAC clones hasbeen established, which extends approximately 3 Mb from the telomere ofthe long arm of chromosome 16. This contig extends beyond the CY2/CY3somatic cell hybrid breakpoint and includes the 2 regions of minimal LOHidentified at the 16q24.3 region in breast cancer samples. To date, asingle gap of unknown size exists in the contig and will be closed byadditional contig extension experiments. The depth of coverage hasallowed the identification of a minimal tiling path of clones which weresubsequently used as templates for gene identification methods such asexon trapping and genomic DNA sequencing.

EXAMPLE 4 Identification of Candidate Breast Cancer Genes by Analysis ofGenomic DNA Sequence

Selected minimal overlapping BAC and PAC clones from the physical mapcontig were sequenced in order to aid in the identification of candidatebreast cancer genes. DNA was prepared from selected clones using a largescale DNA isolation kit (Qiagen). Approximately 25-50 ug of DNA was thensheared by nebulisation (10 psi for 45 seconds) and blunt ended usingstandard methodologies (Sambrook et al., 1989). Samples were then run onan agarose gel in order to isolate DNA in the 2-4 Kb size range. Thesefragments were cleaned from the agarose using QIAquick columns (Qiagen),ligated into pucl8 and used to transform competent DH10B or DH5a E. colicells. DNA was isolated from transformed clones and was sequenced usingvector specific primers on an ABI377 sequencer. Analysis of genomicsequence was performed using PHRED, PHRAP and GAP4 software on a SUNworkstation. To assist in the generation of large contigs of genomicsequence, information present in the high-throughput genomic sequence(htgs) database at NCBI was incorporated into the assembly phase of thesequence analysis. The resultant genomic sequence contigs were maskedfor repeats and analysed using the BLAST algorithm (Altschul et al.,1997) to identify nucleotide and protein homology to sequences in theGenBank non-redundant and EST databases at NCBI. The genomic sequencewas also analysed for predicted gene structure using the GENSCANprogram.

Homologous IMAGE Consortium cDNA clones were purchased from GenomeSystems and were sequenced. These longer stretches of sequence were thencompared to known genes by nucleotide and amino acid sequencecomparisons using the above procedures. Any sequences that are expressedin the breast are considered to be candidate breast cancer genes. Thosegenes whose function could implicate them in the tumourigenic process,as predicted from homology searches with known proteins, were treated asthe most likely candidates. Evidence that a particular candidate is theresponsible gene comes from the identification of defective alleles ofthe gene in affected individuals or from analysis of the expressionlevels of a particular candidate gene in breast cancer samples comparedwith normal control tissues.

EXAMPLE 5 Identification of the BNO1 Sequence

Genomic Sequence Analysis

Sequences from BAC clones mapping close to the CY2/CY3 breakpoint wereassembled and used in BLASTN homology searches of the dbEST database atNCBI (http://www.ncbi.nlm.nih.gov). A large number of cDNA clones wereidentified to be part of the sequence in this region and these could befurther characterised into distinct UniGene clusters.

The human IMAGE cDNA clone 46795, corresponding to the UniGene clusterHs.7970, was sequenced and used in further database homology searches.This identified an overlapping cDNA clone present in the non-redundantdatabase (GenBank accession number AL117444) that extended the sequenceof clone 46795 further 5′. As this additional 5′ sequence was alsopresent in the genomic sequence located 5′ to the 46795 clone sequence,it confirmed that AL117444 most likely belonged to the Hs.7970transcript. To verify this fact, RT-PCR was done.

Briefly, polyA+mRNA from normal mammary gland (Clontech) was initiallyprimed with an oligo-dT primer and reverse transcribed using theOmniScript RT kit (Qiagen) according to manufacturers conditions.Control reactions were included for each RNA template which omittedreverse transcriptase from the cDNA synthesis step. This was todetermine the presence of any genomic DNA contamination in the RNAsamples. The resulting first strand cDNA was PCR amplified using primersAL-1 (specific for AL117444; SEQ ID NO: 20) and 7970-1 (specific for the3′ end of Hs.7970; SEQ ID NO: 21) using the HotStarTaq kit (Qiagen) in a10 ul reaction volume for 35 cycles. Initially, primers to the controlhouse-keeping gene Esterase D (SEQ ID Numbers: 22 and 23) were used in aseparate reaction to confirm the presence of cDNA templates for eachreverse transcription reaction. Primer sequences are shown in Table 1.These experiments confirmed that the AL117444 and IMAGE cDNA clone 46795belonged to the Hs.7970 transcript.

Northern Analysis

To determine the size of the gene corresponding to Hs.7970, a polyA⁺Northern blot obtained from Clontech was probed with a portion of thegene which was generated by PCR using primers BNO1-2 (SEQ ID NO: 24) andBNO1-3 (SEQ ID NO: 25). Table 1 lists the primer sequences used.Hybridisations were conducted in 10 ml of ExpressHyb solution (Clontech)overnight at 65° C. Filters were washed according to manufacturersconditions. FIG. 2 shows the results of the hybridisation. A single bandof approximately 3.6 kb was detected in the mammary gland, testis,ovary, uterus, prostate, stomach, bladder, spinal cord, brain, pancreasand thyroid. Strongest expression of the gene was seen in the brain. Thesize of the mRNA corresponding to Hs.7970 as determined by the Northernhybridisation indicated that additional 5′ sequence needed to beobtained for the gene.

5′ Sequence Identification

To identify additional 5′ sequence for the Hs.7970 transcript, cDNAsequences present in dbEST corresponding to the mouse orthologue wereutilised. The furthest 5′ extending mouse clone (AU080856) included aputative translation start site. Alignment of AU080856 with the humangenomic sequence containing Hs.7970 delineated the corresponding humansequence of this transcript up to an identical translation start site.Additional RT-PCR experiments were conducted which confirmed thepresence of this 5′ sequence in the human Hs.7970 transcript. Inaddition, further dbEST blast searches identified human cDNA clonescontaining the 5′ end of the gene (eg IMAGE clone 3958783).

The RT-PCR experiments also indicated that Hs.7970 exists as analternatively spliced isoform. This variant is due to the inclusion ofan additional in-frame exon (exon 2.5) located between exons 2 and 3.

In combination, these experiments have established that the Hs.7970transcript, termed BNO1, exists as two alternatively spliced isoforms.One isoform is 3,574 bp in length (SEQ ID NO: 1) and is composed of 9exons that span approximately 55 Kb of genomic DNA, while the secondform of BNO1, which contains exon 2.5, is 3,661 bp in length (SEQ ID NO:3). Table 2 shows the genomic structure of the gene indicating the sizeof introns and exons. Analysis of the BNO1 isoforms indicates thatisoform 1 (minus exon 2.5) has an open reading frame of 1,617nucleotides which codes for a protein of 539 amino acids (SEQ ID NO: 2).Isoform 2 (plus exon 2.5) of BNO1 has an open reading frame of 1,704 bpin length and codes for a protein of 568 amino acids (SEQ ID NO: 4).Partial genomic DNA sequences indicating exon/intron junctions for BNO1are set forth in SEQ ID Numbers: 5-11.

EXAMPLE 6 Characteristics of the BNO1 Sequence

Nucleotide Sequence

A large number of human cDNA clones are present in dbEST which representthe BNO1 gene. An observation of the tissues these cDNA clones werederived from indicates that the gene is also expressed in the adrenalgland, blood, colon, germ cells, heart, kidney, liver, lung, muscle,placenta, synovial membrane, tonsil, cervix, lymph tissue and the skin.These tissues are in addition to those shown to express BNO1 fromNorthern analysis (eg mammary gland, testis, ovary, uterus, prostate,stomach, bladder, spinal cord, brain, pancreas and thyroid) and RT-PCRprocedures (eg human mammary gland).

The human BNO1 nucleotide sequence also detects a large number of mousecDNA clones as previously mentioned. In silico BLAST analysis of mousegenomic DNA sequence in the htgs database at NCBI using the human BNO1nucleotide sequence was successful in identifying the mouse BNO1nucleotide (SEQ ID NO: 12) and corresponding amino acid sequence (SEQ IDNO: 13). The amino acid homology between the two genes is as high as 95%(from amino acid 76 in exon 1 to amino acid 369 in exon 8) whichsuggests that the gene is highly conserved between the two species.

Analysis of the human genomic sequence located 3′ to the BNO1 geneidentified the presence of a number of additional UniGene clusters(Hs.130367, Hs.227170 and Hs.87068) running in the same orientation.RT-PCR experiments using a Hs.130367 (130367-1; SEQ ID NO: 26) andHs.87068 (87068-1; SEQ ID NO: 27) specific primer (see Table 1 forprimer sequences) indicated that these two UniGene clusters could belinked. Sequencing of the RT-PCR product also identified the presence ofthe Hs.227170 cluster. Additional RT-PCR experiments using a BNO1specific primer (BNO1-1; SEQ ID NO: 28) in combination with a Hs.130367specific primer (130367-2; SEQ ID NO: 29) established that Hs.130367could also be linked to the BNO1 gene (see Table 1 for primersequences). Therefore, the three UniGene clusters lying 3′ to BNO1 mostlikely represent variants of this gene that contain additional 3′ UTRsequences. The absence of Northern bands corresponding to the size ofthese BNO1 variants suggests that they are rare forms of the gene. SEQID Numbers:14-19 represent the nucleotide sequences of these variants.

Amino Acid Sequence

The amino acid sequence of BNO1 was used for in silico analysis toidentify homologous proteins in order to establish the function of thegene product. Analysis of the BNO1 protein against the Prosite andPfScan databases(http://www.isrec.isb-sib.ch/software/PFSCAN_form.html), showed thatboth splice isoforms of this protein (SEQ ID Numbers: 2 and 4) containan F-box domain at the amino terminal end with a highly significantexpectation value of 5.6e-10. FIG. 3 shows the sequence of the F-box ofBNO1 compared to the consensus F-box sequence.

The F-box is a protein motif of approximately 50 amino acids thatdefines an expanding family of eukaryotic proteins. F-box containingproteins are the substrate-recognition components of the SCFubiquitin-ligase complexes. These complexes contain four components:Skp1, Cullin, Rbx/Roc1/Hrt1, and an F-box protein. The F-box motiftethers the F-box protein to other components of the SCF complex bybinding the core SCF component, Skp1. This motif is generally found inthe amino half of the proteins and is often coupled with other proteindomains in the variable carboxy terminus of the protein. The most commoncarboxy terminal domains include leucine-rich repeats (LRRs) and WD-40domains. There are currently three subdivisions of the F-box proteinfamily based on the type of carboxy terminal motifs present in theprotein sequences. Following the pattern proposed by Cenciarelli et al(1999) and Winston et al (1999), the nomenclature adopted by the HumanGenome Organisation denotes F-boxes that contain LRRs as FBXL, thosecontaining WD repeats as FBXW, and those lacking all knownprotein-interaction domains FBXO. Analysis of the BNO1 sequence failedto identify additional protein motifs present in the gene indicatingthat BNO1 forms part of the FBXO class of F-box proteins.

The ubiquitin-dependant proteasome degradation pathway is an importantmechanism for regulating protein abundance in eukaryotes. A wide varietyof proteins have been shown to be regulated by this mechanism andinclude oncogenes, tumour suppressor genes, transcription factors andother signalling molecules (Hershko and Ciechanover, 1998; Baumeister etal., 1998). These proteins influence a number of important cellularprocesses such as cell-cycle regulation and apoptosis, modulation of theimmune and inflammatory responses, development and differentiation. Thediverse range of proteins and processes that are regulated byubiquitination suggests that pathologies arising from a disruption ofthe ubiquitination process will also be diverse. For example there isprecedence for this in neurodegenerative disorders. Parkin, a proteinmutated in inherited forms of Parkinson's disease, is an E3 ubiquitinligase (Shimura et al., 2000) and in Alzheimer's disease defectiveubiquitination of cerebral proteins has been identified (Lopez Salon etal., 2000).

The ubiquitination process begins with the addition of ubiquitinmoieties (ubiquitination) to target proteins and follows a multi-stepprocess, the end point of which is the proteolysis of polyubiquitinatedsubstrates by a 26S multi-protein complex (Haas and Siepmann, 1997;Hochstrasser, 1996). Ubiquitination of substrates targeted fordegradation requires 3 classes of enzyme: the ubiquitin-activatingenzymes (E1), the ubiquitin-conjugating enzymes (E2) and the ubiquitinligases (E3). The E3 proteins play an integral role in cell cycleprogression. SCF complexes (a class of E3 ligases) have been shown toregulate the G1-S phase transition (reviewed in Peters, 1998). A widevariety of SCF targets have been reported that include G1-phase cyclins,cyclin-dependant kinase inhibitors, DNA replication factors,transcription factors that promote cell-cycle progression and otherimportant cellular proteins. The sequences present in the variablecarboxy terminal region of the F-box proteins therefore allowrecruitment of specific substrates for ubiquitination and subsequentdegradation.

Recent studies of the Von Hippel-Lindau (VHL) tumour suppressor proteinhave shown that it is part of a complex that functions as aubiquitin-protein ligase E3 (Zaibo et al., 2001). The VHL protein linksthe ligase complex to target proteins which include HIFα (hypoxiainducible factor) (Ohh et al., 2000; Cockman et al., 2000) and VDU1 (VHLinteracting deubiquitinating enzyme 1) (Zaibo et al., 2001). HIFα hasbeen shown to regulate genes involved in angiogenesis, a processcritical for the growth of tumours (Wang et al., 1995; Semenza, 2000),while VDU1 has deubiquitinating activity.

The predicted role of BNO1, based on the presence of the F-box domain,indicates that the gene may be involved in a diverse range of cellularprocesses including cell-cycle regulation. Combined with the fact thatBNO1 lies in a region of LOH seen in breast and other tumour typessuggests BNO1 is an ideal candidate breast cancer gene.

EXAMPLE 7 Examination of the Expression Level of BNO1 in Breast CancerCell Lines

To investigate a potential role of BNO1 in breast cancer, the level ofexpression of the gene was compared in breast cancer cell lines withnormal tissue controls. Examination of the genomic sequence surroundingBNO1 shows that the 5′ end including exon 1 is extremely G-C richsuggesting the presence of a CpG island. While not wishing to be boundby theory, this raises the possibility that epigenetic mechanisms toinactivate BNO1 function may exist. Abnormal methylation at this sitemay result in a down-regulation of BNO1 transcription of the remainingcopy of the gene. Recent studies have shown that this mechanism has beenresponsible for the inactivation of other tumour suppressor genes suchas RB1 (Ohtani-Fujita et al., 1997), VHL (Prowse et al., 1997), MLH1(Herman et al., 1998) and BRCA1 (Esteller et al., 2000).

To detect the level of expression of BNO1 in cancer samples comparedwith normal controls, quantitative RT-PCR using BNO1 specific primerswas done. This initially involved the isolation of RNA from breastcancer cell lines along with appropriate cell line controls.

Breast/Prostate Cancer Cell Lines and RNA Extraction

Cancer cell lines were purchased from ATCC (USA) and grown in therecommended tissue culture medium. Breast cancer cell lines were chosenfor RT-PCR analysis that demonstrated homozygosity for a number ofmarkers mapping to chromosome 16q indicating potential LOH for thischromosomal arm. Cells were harvested from confluent cultures and totalRNA was extracted using the RNAeasy kit (Qiagen). Breast cancer celllines obtained for RNA extraction were BT549, MDA-MB-468, CAMA-1,ZR75-30, MDA-MB-157, ZR75-1, SKBR3, MDA-MB-231, T47D, and MDA-MB-436.The normal breast epithelial cell line MCF12A and the prostate cancercell line PC3 were also purchased. PolyA⁺ mRNA was subsequently isolatedfrom all sources using the Oligotex bead system (Qiagen). PolyA⁺ mRNAfrom normal mammary gland, prostate, ovary and liver was purchasedcommercially (Clontech, USA).

Reverse Transcription

PolyA⁺ mRNA was primed with oligo-dT primers and reverse transcribedusing the Omniscript RT kit (Qiagen) according to manufacturersconditions. Control reactions were included for each RNA template whichomitted reverse transcriptase from the cDNA synthesis step. This was todetermine the presence of any genomic DNA contamination in the RNAsamples.

cDNA Normalisation

Internal standard curve amplicons were generated from a mixed pool ofnormal tissue cDNA using the HotStarTaq™ DNA Polymerase kit (Qiagen). Areaction mix sufficient to generate >1 ug of amplicon cDNA contained 10ul of 10×PCR buffer (containing 15 MM MgCl₂), 2 ul of 10 mM DNTP mix,0.5 uM of each primer, 0.5 ul of 2.5 units HotStarTaq polymerase(Qiagen), 100 ng of cDNA template and DEPC treated water to 100 ul.Amplification cycling was performed as follows: 94° C. for 10 minutesfollowed by 35 cycles at 93° C. for 20 seconds, 60° C. for 30 secondsand 70° C. for 30 seconds with a final extension at 72° C. for 4minutes. Amplicons were purified using the QIAquick gel extraction kit(Qiagen) according to manufacturers conditions and concentrations weremeasured at A₂₆₀. Purified amplicons were serially diluted 10-fold from10 ng/ul to 1 fg/ul. These dilutions served as internal standards ofknown concentration for real-time analysis of BNO1 specific amplicons asdescribed below.

Real-time PCR

All cDNA templates were amplified using the SYBR Green I PCR Master Mixkit (PE Biosystems, USA). PCR reactions were in a volume of 25 ul andincluded 12.5 ul of SYBR Green I PCR Master mix, 0.5 uM of each primer,2 ul normalised cDNA template (see below) and 9.5 ul of water. Real-timePCR analysis was performed using the Rotor-Gene™2000 (Corbett Research,AUS) with the following amplification cycling conditions: 94° C. for 10minutes followed by 45 cycles of 93° C. for 20 sec, 60° C. for 30 secand 70° C. for 30 sec. Fluorescence data was acquired at 510 nm duringthe 72° C. extension phase. Melt curve analyses were performed with aninitial 99-50° C. cycling followed by fluorescence monitoring duringheating at 0.2° C./second to 99° C. Prior to real-time quantification,product size and specificity was confirmed by ethidium bromide stainingof 2.5% agarose gels following electrophoresis of completed PCRs.Control and BNO1 specific primers used for all real-time PCRapplications are listed in Table 1 and are represented by the SEQ IDNumbers: 30-41.

Real-time PCR Quantification

Quantification analyses were performed on the Rotor-Gene™ DNA sampleanalysis system (Version 4.2, Build 96). Standard curves were generatedby amplifying 10-fold serial dilutions (1 ul of 10 ρg/ul down to 1 ul of1 fg/ul in triplicate) of the internal standard amplicon duringreal-time PCR of BNO1 amplicons from normal tissues and breast cancercell lines. Internal standard amplicon concentrations were arbitrarilyset to 1.0e+12 copies for 10 ρg standards to 1.0e+08 copies for 1 fgstandards. C_(T) (cycle threshold) coefficients of variation for allinternal standard dilutions averaged 2% between triplicate sampleswithin the same and different runs. The Rotor-Gene™ quantificationsoftware generated a line of best-fit at the parameter C_(T) anddetermined unknown normal tissue and breast cancer cell line BNO1amplicon copy numbers by interpolating the noise-band intercept of BNO1amplicons against the internal standards with known copy numbers.

Normalization and Relative Expression of Data

To account for variation in sample-to-sample starting templateconcentrations, RiboGreen™ RNA quantitation (Molecular Probes) was usedto accurately assay 1 ug of normal tissue and breast cancer cell lineRNA for cDNA synthesis. Selected housekeeping gene expression levelswere then analyzed in all samples to determine the most accurateendogenous control for data normalization. Housekeeping ampliconsincluded Esterase D (Accession Number M13450), Cyclophilin (AccessionNumber X52851), APRT (Accession Number M16446) and RNA Polymerase II(Accession Number Z47727). As Cyclophilin displayed the least variableexpression profile, calculated BNO1 copy numbers were divided by therespective Cyclophilin amplicon copy number for each breast cancer cellline and normal tissue analyzed. BNO1 copy numbers in normalized normalbreast cDNA were arbitrarily set to a ‘baseline’ of 1.e+06 copies.Breast cancer cell lines and other normal tissue cDNA copy numbers werecalculated relative to the ‘baseline’. Data was expressed as logrelative mRNA copy number. FIG. 4 shows the results from theseexperiments.

The degree of variation in mRNA expression levels for Cyclophilin, RNApolymerase II subunit and APRT were relatively uniform between thenormal tissues and cancer cell lines. Three-way combinations fornormalization between Cyclophilin, RNA polymerase II subunit and APRTdemonstrated a mean 7-fold and maximum 50-fold variance in mRNAexpression level between samples. The significance of variable mRNAexpression levels within a gene of interest may therefore reasonably beevaluated based on these normalization results. A predicted aberrantdecrease in gene of interest mRNA copy number of ˜100 fold in breastcancer cell lines relative to a ‘baseline’ normal breast expressionlevel was therefore considered to be significantly abnormal.

FIG. 4 indicates that BNO1 amplicons specific for exon 5-7 and isoform 1(minus exon 2.5) show a consistent pattern of mRNA expression amongnormal tissues and breast cancer cell lines. For both ampliconsanalyzed, the breast cancer cell lines MDA-MB-468, SK-BR3, MDA-MB-231and the prostate cancer cell line PC3 all display low-level mRNAexpression with respect to the ‘baseline’ normal breast tissue. Asignificant 725-fold reduction in BNO1 exon 5-7 mRNA expression wasdetected in SK-BR3 with respect to the normal breast tissue expression(equivalent to an approximately 350,000-480,000 down-regulation in mRNAmolecule expression). Similar results were obtained for isoform 1 ofBNO1 (minus exon 2.5), with a 248-fold reduction in mRNA expression inSK-BR3 (equivalent to an approximately 300,000-1,000,000 down-regulationin mRNA molecule expression). BNO1 isoform 2 (plus exon 2.5) displayedsignificantly low mRNA expression in the cell lines MDA-MB-468, CAMA-1,SK-BR3 and MDA-MB-231, with no expression detected in ZR75-30. Theseresults indicate that both isoforms of the BNO1 gene are down-regulatedin certain breast cancer cell lines as well as a prostate cancer cellline. The exact mechanism of this down-regulation is not known at thisstage but may result from mechanisms such as mutation or promotermethylation. From these expression studies we propose that BNO1 is aprotein responsible for the development of breast and prostate cancer.Due to its broad tissue expression pattern, BNO1 may also be implicatedin cancers originating from other tissues.

Other methods to detect BNO1 expression levels may be used. Theseinclude the generation of polyclonal or monoclonal antibodies, which areable to detect relative amounts of both normal and mutant forms of BNO1using various immunoassays such as ELISA assays (See Example 11 and 12).

EXAMPLE 8 Analysis of Tumours and Cell Lines for BNO1 Mutations

The BNO1 gene was screened by SSCP analysis in DNA isolated from tumoursfrom series 1 as well as a subset of series 2 tumours (not shown inFIG. 1) that displayed loss of the whole long arm of chromosome 16.These samples from series 2 were used due to larger amounts of DNA beingavailable. In total 45 primary breast tumours with 16q LOH were examinedfor mutations.

A number of cell lines were also screened for mutations. These included22 breast cancer cell lines (BT20, BT474, BT483, BT549, CAMA-1, DU4475,Hs578T, MCF7, MB157, MB231, MB361, MB415, MB436, MB453, MB468, SKBR3,T47D, UACC893, ZR75-1, ZR75-30, MB134 and MB175), 2 prostate cancer celllines (LNCAP and PC3), 2 gastric carcinoma cell lines (AGS and KATO), 1liver cancer cell line (HEP2) and 2 normal breast epithelial cell lines(HBL100 and MCF12A). All cell lines were purchased from ATCC, grownaccording to manufacturers conditions, and DNA isolated from culturedcells using standard protocols (Wyman and White, 1980; Sambrook et al.,1989).

BNO1 exons were amplified by PCR using flanking intronic primers, whichwere labeled at their 5′ ends with HEX. An exception was made for exon 1and 8, as due to their size had to be split into 2 overlappingamplimers. Table 3 lists the sequences of all primers used for the SSCPanalysis, the expected amplimer size and the MgCl₂ concentration used inthe PCR reaction. Typical PCR reactions were performed in 96-well platesin a volume of 10 ul using 30 ng of template DNA. Cycling conditionswere an initial denaturation step at 94° C. for 3 minutes followed by 35cycles of 94° C. for 30 seconds, 60° C. for 1^(1/2) minutes and 72° C.for 1^(1/2) minutes. A final extension step of 72° C. for 10 minutesfollowed. Twenty ul of loading dye comprising 50% (v/v) formamide, 12.5mM EDTA and 0.02% (w/v) bromophenol blue were added to completedreactions which were subsequently run on 4% polyacrylamide gels andanalysed on the GelScan 2000 system (Corbett Research, AUS) according tomanufacturers specifications.

Of all 12 amplicons tested, only 2 identified SSCP bandshifts. In exon2.5, identical bandshifts were seen in 2 tumour samples from series 1(380 and 355) and the breast cancer cell line MCF7. SSCP analysis of thecorresponding normal DNA from sample 380 and 355 identified the samebandshift indicating the change was most likely not causative for thedisease. Sequence analysis of this bandshift in all samples showed thata single nucleotide base change (−5T→C) was responsible for thisbandshift. This change does not affect the consensus splice acceptorsite score for this exon and hence most likely represents apolymorphism. The incidence of this change in the general population hasnot been examined as yet. In exon 8b, a bandshift was identified in onlya single cancer cell line (KATO). Sequencing of this bandshift indicateda C→T change at position +10 of this amplicon which is located in thesplice donor site (5′ splice site). This base change occurs outside thesplice junction consensus sequence and it is envisaged that the mutationhas no effect on splicing of this exon.

EXAMPLE 9: Immunoprecipitation of BNO1 and Skp1

To test if BNO1 contained a functional F-box motif, aco-immunoprecipitation assay was employed. This involved cloning of thefull-length Myc-tagged open reading frame of BNO1 into the SalI/ClaIsites of the retroviral expression vector LNCX2 (Clontech) usingstandard techniques (Sambrook et al., 1989). Following this, 10⁷ 293Tcells were transfected with 10 ug of the BNO1-LNCX2 construct orseparately with LCNX2 vector alone as a control using Lipofectamine 2000(Invitrogen) according to manufacturers instructions. Cells wereharvested 24 hours post-transfection and lysed in 2 ml of lysis buffer(50 mM Tris-HCL [pH 7.5], 150 mM NaCl, 0.5% Nonidet P-40 supplementedwith 1 mM PMSF and 5 μg/ml leupeptin, antipain and aprotenin). Followingthis, 0.5 ml of the cell lysate was incubated with 2 ug of anti-Mycmonoclonal antibody (Roche) or anti-p19^(SkP1) rabbit polyclonalantibody (Neo Markers, Fremont, Calif.) for 1 hour and proteinA-Sepharose for 1 hour at 4° C. Immune complexes were washed three timeswith 1 ml of lysis buffer followed by separation on 10% SDS-PAGE andimmunoblotting according to standard techniques (Sambrook et al., 1989).

Results from these experiments indicated that BNO1 specificallyco-precipitated with endogenous Skp1, confirming both an associationbetween these two proteins and the presence of a functional F-box withinBNO1. This interaction indicates that BNO1 belongs to a novelE3-ubiquitin ligase complex that may be critical for the controlleddegradation of BNO1 specific substrates.

EXAMPLE 10 Analysis of the BNO1 gene

The following methods are used to determine the structure and functionof BNO1.

Biological Studies

Mammalian expression vectors containing BNO1 cDNA (representing bothisoforms of BNO1) can be transfected into breast, prostate or othercarcinoma cell lines that have lesions in the gene. Phenotypic reversionin cultures (eg cell morphology, growth of transformants in soft-agar,growth rate) and in animals (eg tumourigenicity in nude mice) isexamined. These studies can utilise wild-type or mutant forms of BNO1.Deletion and missense mutants of BNO1 can be constructed by in vitromutagenesis.

Molecular Biological Studies

The ability of both isoforms of the BNO1 protein to bind known andunknown proteins can be examined. Procedures such as the yeasttwo-hybrid system are used to discover and identify any functionalpartners, particularly BNO1 specific substrates or isoform-specificsubstrates that are targeted for degradation by ubiquitination. Theprinciple behind the yeast two-hybrid procedure is that many eukaryotictranscriptional activators, including those in yeast, consist of twodiscrete modular domains. The first is a DNA-binding domain that bindsto a specific promoter sequence and the second is an activation domainthat directs the RNA polymerase II complex to transcribe the genedownstream of the DNA binding site. Both domains are required fortranscriptional activation as neither domain can activate transcriptionon its own. In the yeast two-hybrid procedure, the gene of interest orparts thereof (BAIT), is cloned in such a way that it is expressed as afusion to a peptide that has a DNA binding domain. A second gene, ornumber of genes, such as those from a cDNA library (TARGET), is clonedso that it is expressed as a fusion to an activation domain. Interactionof the protein of interest with its binding partner brings theDNA-binding peptide together with the activation domain and initiatestranscription of the reporter genes. The first reporter gene will selectfor yeast cells that contain interacting proteins (this reporter isusually a nutritional gene required for growth on selective media). Thesecond reporter is used for confirmation and while being expressed inresponse to interacting proteins it is usually not required for growth.

The nature of the BNO1 interacting genes and proteins can also bestudied such that these partners can also be targets for drug discovery.Of particular interest are those BNO1-interacting proteins that aretargeted for ubiquitination and subsequent degradation by theBNO1-containing ubiquitin-E3 ligase.

Structural Studies

BNO1 recombinant proteins can be produced in bacterial, yeast, insectand/or mammalian cells and used in crystallographical and NMR studies.Together with molecular modeling of the protein, structure-driven drugdesign can be facilitated.

EXAMPLE 11 Generation of Polyclonal Antibodies Against BNO1

The knowledge of the nucleotide and amino acid sequence of BNO1 allowsfor the production of antibodies, which selectively bind to BNO1 proteinor fragments thereof. Following the identification of mutations in thegene, antibodies can also be made to selectively bind and distinguishmutant from normal protein. Antibodies specific for mutagenised epitopesare especially useful in cell culture assays to screen for malignantcells at different stages of malignant development. These antibodies mayalso be used to screen malignant cells, which have been treated withpharmaceutical agents to evaluate the therapeutic potential of theagent.

To prepare polyclonal antibodies, short peptides can be designedhomologous to the BNO1 amino acid sequence. Such peptides are typically10 to 15 amino acids in length. These peptides should be designed inregions of least homology to the mouse orthologue to avoid cross speciesinteractions in further down-stream experiments such as monoclonalantibody production. Synthetic peptides can then be conjugated to biotin(Sulfo-NHS-LC Biotin) using standard protocols supplied withcommercially available kits such as the PIERCE ™ kit (PIERCE).Biotinylated peptides are subsequently complexed with avidin in solutionand for each peptide complex, 2 rabbits are immunized with 4 doses ofantigen (200 μg per dose) in intervals of three weeks between doses. Theinitial dose is mixed with Freund's Complete adjuvant while subsequentdoses are combined with Freund's Immuno-adjuvant. After completion ofthe immunization, rabbits are test bled and reactivity of sera assayedby dot blot with serial dilutions of the original peptides. If rabbitsshow significant reactivity compared with pre-immune sera, they are thensacrificed and the blood collected such that immune sera can separatedfor further experiments.

EXAMPLE 12 Generation of Monoclonal Antibodies Specific for BNO1

Monoclonal antibodies can be prepared for BNO1 in the following manner.Immunogen comprising intact BNO1 protein or BNO1 peptides (wild type ormutant) is injected in Freund's adjuvant into mice with each mousereceiving four injections of 10 to 100 ug of immunogen. After the fourthinjection blood samples taken from the mice are examined for thepresence of antibody to the immunogen. Immune mice are sacrificed, theirspleens removed and single cell suspensions are prepared (Harlow andLane, 1988). The spleen cells serve as a source of lymphocytes, whichare then fused with a permanently growing myeloma partner cell (Kohlerand Milstein, 1975). Cells are plated at a density of 2×10⁵ cells/wellin 96 well plates and individual wells are examined for growth. Thesewells are then tested for the presence of BNO1 specific antibodies byELISA or RIA using wild type or mutant BNO1 target protein. Cells inpositive wells are expanded and subcloned to establish and confirmmonoclonality. Clones with the desired specificity are expanded andgrown as ascites in mice followed by purification using affinitychromatography using Protein A Sepharose, ion-exchange chromatography orvariations and combinations of these techniques.

Disclosed in one embodiment is an isolated gene comprising thenucleotide sequence set forth in SEQ ID NO: 1 from base 4 to base 1,621or set forth in SEQ ID NO: 3 from base 4 to base 1,708 and BNO1 controlelements. The BNO1 control elements can be those which mediateexpression in breast, prostate, liver and ovarian tissue.

In another embodiment, provided is a cell transformed with an expressionvector as disclosed herein. In the cell, BNO1 can be expressed in amutant form. In the cell, BNO1 expression can switched off. The cell canbe an eukaryotic cell. In one embodiment a method of preparing apolypeptide is provided, wherein the method comprising the steps of:culturing such cells under conditions effective for production of thepolypeptide; and harvesting the polypeptide. Also disclosed is a methodof screening for drug candidates, comprising the steps of: providing acell as described above; adding a drug candidate to the cell; anddetermining the effect of the drug candidate on the expression of BNO1by the cell. The use of a cell as described above in the screening ofdrug candidates is also disclosed, as is the use of a nucleic acid asdescribed herein in screening for drug candidates.

Also disclosed is an isolated polypeptide comprising the amino acidsequence set forth in SEQ ID Numbers: 2 or 4. In another embodiment,disclosed is an isolated polypeptide, comprising the amino acid sequenceset forth in SEQ ID Numbers: 2 or 4, or a fragment thereof, capable offorming part of a ubiquitin-ligase complex involved in proteindegradation through ubiquitination. In another embodiment, disclosed isan isolated polypeptide capable of forming part of a ubiquitin-ligasecomplex involved in protein degradation through ubiquitination that hasat least 70% identity with the amino acid sequence set forth in SEQ IDNumbers: 2 or 4; optionally, with at least 85% sequence identity, andalso optionally, with at least 95% sequence identity. The sequenceidentity can determined using the BLASTP algorithm and the BLOSSUM62default matrix. An isolated polypeptide consisting of the amino acidsequence set forth in SEQ ID Numbers: 2 or 4 is also disclosed, as is anisolated polypeptide as described herein which is inactivated as aresult of mutation or polymorphism.

An antibody that binds a polypeptide as disclosed herein is alsoprovided. The antibody can be selected from the group consisting of amonoclonal antibody, a humanised antibody, a chimaeric antibody or anantibody fragment including a Fab fragment, F(ab′)₂ fragment, Fvfragment, single chain antibodies and single domain antibodies.

In another embodiment disclosed is a method of treatment of a disorderassociated with decreased expression or activity of BNO1, comprisingadministering an isolated nucleic acid molecule as defined herein to asubject in need of such treatment. An expression vector comprising theisolated nucleic acid molecule operably linked to suitable controlelements can be administered. The use of a nucleic acid molecule asdisclosed herein in the manufacture of a medicament for the treatment ofdisorder associated with decreased expression or activity of BNO1 isalso provided. Optionally, the nucleic acid molecule is a part of anexpression vector which also includes suitable control elements.

Also disclosed is a method for the treatment of a disorder associatedwith decreased expression or activity of BNO1, comprising administeringa compound which increases expression or activity of BNO1 to a subjectin need of such treatment.

Also disclosed is a method for the treatment of a disorder associatedwith increased expression or activity of BNO1, comprising administeringan antagonist of BNO1 to a subject in need of such treatment. Theantagonist of BNO1 can be an antibody as defined herein. Also disclosedis the use of an antagonist of BNO1 in the manufacture of a medicamentfor the treatment of a disorder associated with increased expression oractivity of BNO1. The antagonist of BNO1 can be an antibody as definedherein.

Also disclosed is a method for the treatment of a disorder associatedwith increased expression or activity of BNO1, comprising administeringa nucleic acid molecule which is the complement of any one of thenucleic acid molecules as defined herein, the transcription product ofwhich is a RNA molecule that hybridizes with the mRNA encoded by BNO1.Also disclosed is the use of an isolated nucleic acid molecule which isthe complement of a nucleic acid molecule as defined herein, thetranscription product of which is a mRNA that hybridizes with the mRNAencoded by BNO1, in the manufacture of a medicament for the treatment ofa disorder associated with increased activity or expression of BNO1.

Also disclosed is a method for the treatment of a disorder associatedwith increased expression or activity of BNO1, comprising administeringa compound which decreases expression or activity of BNO1 to a subjectin need of such treatment. The use of a compound which modulates theexpression of BNO1 in the preparation of a medicament for the treatmentof disorders associated with abnormal expression or activity of BNO1 isalso disclosed.

Also disclosed are a pharmaceutical composition comprising a nucleicacid molecule as defined herein, and a pharmaceutical acceptablecarrier; a pharmaceutical composition comprising an antibody as definedherein, and a pharmaceutical acceptable carrier; and a pharmaceuticalcomposition comprising a compound that modulates the expression of BNO1,and a pharmaceutical acceptable carrier.

Also disclosed is a method for screening for a compound capable ofmodulating the activity of BNO1 comprising combining a peptide asdefined herein and a candidate compound, and determining the binding ofthe candidate compound to the peptide. The use of a peptide as definedherein in screening for candidate pharmaceutical agents is alsodisclosed.

Also disclosed is a method for the diagnosis of a disorder associatedwith mutations in BNO1, or a predisposition to such disorders in apatient, comprising the steps of: obtaining a sample which includes BNO1or a nucleic acid which codes for BNO1 from the patient; comparing BNO1or a nucleic acid which codes for TSG18 from the sample with wild-typeBNO1 or a nucleic acid which codes for it in order to establish whetherthe person expresses a mutant BNO1. Optionally, the nucleotide sequenceof DNA from the patient is compared to the sequence of DNA encodingwild-type BNO1.

Also disclosed is a method for the diagnosis of a disorder associatedwith abnormal expression or activity of BNO1, or a predisposition tosuch disorders, comprising the steps of: establishing a profile fornormal expression of BNO1 in unaffected subjects; measuring the level ofexpression of BNO1 in a person suspected of abnormal expression oractivity of BNO1; and comparing the measured level of expression withthe profile for normal expression. Reverse transcriptase PCR can beemployed to measure levels of expression. A hybridisation assay using aprobe derived from BNO1, or a fragment thereof, can be employed tomeasure levels of expression. The probe can have at least 50% sequenceidentity to a nucleotide sequence encoding BNO1, or a fragment thereof.

Also disclosed is a method for the diagnosis of a disorder associatedwith BNO1, or a predisposition to such disorders, comprising the stepsof: establishing a physical property of wild-type BNO1; obtaining BNO1from a person suspected of an abnormality of BNO1; and measuring theproperty for the BNO1 expressed by the person and comparing it to theestablished property for wild-type BNO1 in order to establish whetherthe person expresses a mutant BNO1. The property can be theelectrophoretic mobility. The property can be the proteolytic cleavagepattern.

Also disclosed is a genetically modified non-human animal transformedwith an isolated nucleic acid molecule as defined herein. Thegenetically modified non-human animal can be selected from the groupconsisting of rats, mice, hamsters, guinea pigs, rabbits, dogs, cats,goats, sheep, pigs and non-human primates such as monkeys andchimpanzees. In one embodiment, the genetically modified non-humananimal is a mouse. In one embodiment, the genetically modified non-humananimal is an animal in which BNO1 gene function has been knocked out. Inthis embodiment, the genetically modified non-human animal can beselected from the group consisting of rats, mice, hamsters, guinea pigs,rabbits, dogs, cats, goats, sheep, pigs and non-human primates such asmonkeys and chimpanzees. In one embodiment the genetically modifiednon-human animal is a mouse. The use of a genetically modified non-humananimal as defined herein in screening for candidate pharmaceuticalcompounds is also disclosed.

Also disclosed is a microarray comprising a nucleic acid encoding eitherisoform of BNO1 or, a fragment thereof, or nucleic acids encoding bothisoforms of BNO1, or fragments thereof. The use of either isoform ofBNO1 in order to identify BNO1-specific protein substrates that aretargeted for degradation by ubiquitination is also disclosed.

Also disclosed is a complex of BNO1 and a BNO1-specific proteinsubstrate, as is the use of a complex of BNO1 and a BNO1-specificprotein substrate in the screening for candidate pharmaceuticalcompounds.

Also disclosed is a complex of BNO1 and proteins of the ubiquitin-E3ligase complex, as is the use of a complex of BNO1 and proteins of theubiquitin-E3 ligase complex in screening for candidate pharmaceuticalcompounds.

Also disclosed is a mutant BNO1 polypeptide which cannot form a complexwith its specific protein substrate, as is the use of a mutant BNO1polypeptide as defined herein in screening for candidate pharmaceuticalcompounds. A mutant BNO1 polypeptide which cannot form a complex withthe ubiquitin-E3 ligase complex is disclosed, as is the use of a mutantBNO1 polypeptide as defined herein in screening for candidatepharmaceutical compounds.

Also disclosed is an isolated nucleic acid molecule comprising thepartial genomic DNA sequences set forth in any one of SEQ ID Numbers:5-11. Also disclosed is an isolated nucleic acid molecule comprising thenucleotide sequence set forth in SEQ ID NO: 12. Also disclosed is anisolated polypeptide comprising the amino acid sequence set forth in SEQID NO: 13. Also disclosed is an isolated nucleic acid comprising thenucleotide sequence set forth in any one of SEQ ID Numbers: 14-19.

INDUSTRIAL APPLICABILITY

The BNO1 gene is implicated in cancer and based on its role in theubiquitination process, BNO1 may also be implicated in cellularmechanisms which are regulated by this process. The novel DNA moleculesof the present invention are therefore useful in methods for the earlydetection of disease susceptible individuals as well as in therapeuticprocedures associated with these disease states. TABLE 1 Primers Usedfor Analysis of BNO1 Primer Name Primer Sequence (5′ → 3′) AL-1 GTA AAGAAG GAT GAG TTC TCC (SEQ ID NO: 20) 7970-1 AGC TGA GCA TCA CAA TCT CC(SEQ ID NO: 21) ESTD-F GGA GCT TCC CCA ACT CAT AAA TGC C (SEQ ID NO: 22)ESTD-R GCA TGA TGT CTG ATG TGG TCA GTA A (SEQ ID NO: 23) BNO1-2 TGC GAAGCT GCT TCA CCG AT (SEQ ID NO: 24) BNO1-3 GGC CGT ACA TGC ACT CCA CTG(SEQ ID NO: 25) 130367-1 GAG AAC CTG CAG TTG TGC TG (SEQ ID NO: 26)87068-1 ATG GTG CTG CTT GTA GCA AG (SEQ ID NO: 27) 130367-2 ACA CTC AGCAGT GGA CAC TTG (SEQ ID NO: 28) Cyclophilin F¹ GGC AAA TGC TGG ACC CAACAC AAA (SEQ ID NO: 30) Cyclophilin R¹ CTA GGC ATG GGA GGG AAC AAG GAA(SEQ ID NO: 31) APRT-F¹ GAC TGG GCT GCG TGC TCA TCC (SEQ ID NO: 32)APRT-R¹ AGG CCC TGT GGT CAC TCA TAC TGC (SEQ ID NO: 33) RNA PolymeraseII-F¹ AGG GGC TAA CAA TGG ACA CC (SEQ ID NO: 34) RNA Polymerase II-R¹CCG AAG ATA AGG GGG AAC TAC T (SEQ ID NO: 35) BNO1 (Exon 5-7)-F¹ CCG GCGGGA GGC AGG AGG AGT (SEQ ID NO: 36) BNO1 (Exon 5-7)-R¹ GCG GCG GTA GGTCAG GCA GTT GTC (SEQ ID NO: 37) BNO1 (Isoform 1)-F¹ TGC GAA GCT GCT TCACCG AT (SEQ ID NO: 38) BNO1 (Isoform 1)-R¹ GGC CGT ACA TGC ACT CCA CTG(SEQ ID NO: 39) BNO1 (Isoform 2)-F¹ GTG AAG TCG GGA CGT TTT GTG A (SEQID NO: 40) BNO1 (Isoform 2)-R¹ CCG TGG TGG GGC CCT TTG TGG (SEQ ID NO:41)Note:¹These primers were labeled at their 5′ ends with HEX. Isoform 1 of BNO1lacks exon 2.5 (SEQ ID NO:1). Isoform 2 of BNO1 contains exon 2.5 (SEQID NO:3)

TABLE 2 Splice Sites of the BNO1 Gene Consensus Consensus Intron Size 3′Splice site strength 5′ Splice site strength size Exon (bp)(intron/exon) (%) (exon/intron) (%) (bp) 1 343 5′UTRTGCCGTGAGG/gtgagcgcgc 83.03 23042 (SEQ ID NO: 51) 2 72cttgttacag/AGTATGGTGT 94.28 TATGCGAAGC/gtgagtgaat 75.36 1797 (SEQ ID NO:42) (SEQ ID NO: 52) 2.5 87 gtctgttcag/GTATAAACCC 90.0TACACCTGCC/gtatgtacct 66.97 11160 (SEQ ID NO: 43) (SEQ ID NO: 53) 3 77cctcctgtag/TGCTTCACCG 78.70 GAACGTGGTG/gtaagtcccg 92.15 3408 (SEQ ID NO:44) (SEQ ID NO: 54) 4 168 cctcctgtag/GTGGACGGCC 84.95CCACATCCAG/gtgtgtgcag 85.40 646 (SEQ ID NO: 45) (SEQ ID NO: 55) 5 75aacactgaag/ATTGTGAAGA 63.39 GAGGCAGGAG/gtgagcccac 90.87 6612 (SEQ ID NO:46) (SEQ ID NO: 56) 6 110 cttttggaag/GAGTTTCGGA 85.65GTCACTACGA/gtgagtgcgg 76.46 697 (SEQ ID NO: 47) (SEQ ID NO: 57) 7 154ctccccacag/CAACTGCCTG 85.32 CAAGATCACG/gtgagtggcg 88.50 1017 (SEQ ID NO:48) (SEQ ID NO: 58) 8 401 tgctccacag/GGCGACCCCA 89.22GCAGGATGTG/gtaaggatg 87.59 2375 (SEQ ID NO: 49) (SEQ ID NO: 59) 9 2174ttctgctcag/TTTTTATGGC 90.62 3′UTR (SEQ ID NO: 50)

TABLE 3 Primers used for the SSCP analysis of BNO1 Primer 1 Primer 2Product Exon (5′ → 3′) (5′ → 3′) [MgCl_(s)] Size (bp)  1aGCGCTGGAGCGTGCGCACA AGCTCGGGCGGCAGCTCCA 2.0 mM 269 (SEQ ID NO: 60) (SEQID NO: 72)  1b GGTCGGGGGCGGCTTGTG GCCTCCACCTGGCAGGGA 2.0 mM 252 (SEQ IDNO: 61) (SEQ ID NO: 73) 2 CTGTCGCGTTATGAGTTGTTG GTACAAAGTTAATCATGGATGGT2.0 mM 168 (SEQ ID NO: 62) (SEQ ID NO: 74)   2.5 AGGCATTGGGTCGTATTCACAGAAGCCAAAGCTCGCAGGA 1.5 mM 198 (SEQ ID NO: 63) (SEQ ID NO: 75) 3GGCACGCTGGGTCTAACAC CCTGCCCGTGCACAGACCT 1.5 mM 167 (SEQ ID NO: 64) (SEQID NO: 76) 4 CTCATGGACCTTTGCCCATCT GTCTGCAGCTGAGAATAGCAC 1.0 mM 290 (SEQID NO: 65) (SEQ ID NO: 77) 5 GTGATGGACTCTGTTCCTCAC AGGTCCGCACCATATGAACAC2.0 mM 170 (SEQ ID NO: 66) (SEQ ID NO: 78) 6 CACAGCCTCCTGTCATATGGAACCCCAGCACCGAGCAGGA 1.5 mM 187 (SEQ ID NO: 67) (SEQ ID NO: 79) 7GGCGTTCTCAGTCCTGCCT CCCTGACTCCACAGCCCAC 1.5 mM 284 (SEQ ID NO: 68) (SEQID NO: 80)  8a CTGGCCTGAGCCCTGCTGA ACCCTCTCGCGCACCTCCA 1.0 mM 171 (SEQID NO: 69) (SEQ ID NO: 81)  8b CAATGAGCTCTCCCGCATC CCATGCTGTCCCACCTTCA1.5 mM 354 (SEQ ID NO: 70) (SEQ ID NO: 82) 9 AGAATGCTGTACGTGGCGTGAGGAGGTGAGGGACTGAATG 1.0 mM 292 (SEQ ID NO: 71) (SEQ ID NO: 83)Note:All primes were labelled at their 5′ ends with HEX.

REFERENCES

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1-88. (canceled)
 89. An isolated polypeptide comprising the amino acidsequence set forth in SEQ ID Nos: 2 or
 4. 90. An isolated polypeptide,comprising the amino acid sequence set forth in SEQ ID NOs: 2 or 4,capable of forming part of a ubiquitin-ligase complex involved inprotein degradation through ubiquitination.
 91. An isolated polypeptidecapable of forming part of a ubiquitin-ligase complex involved inprotein degradation through ubiquitination which has at least 95%sequence identity with the amino acid sequence set forth in SEQ ID NOs:2 or
 4. 92. An isolated polypeptide consisting of the amino acidsequence set forth in SEQ ID NOs: 2 or
 4. 93. A method for screening forcandidate pharmaceutical agents for the treatment of breast cancer, themethod comprising combining a polypeptide as claimed in any one ofclaims 89 to 92 and a candidate compound, and determining the binding ofsaid candidate compound to said polypeptide.
 94. A method of diagnosingbreast cancer, the method comprising detecting the polypeptide of anyone of claims 89 to 92 in a biological sample suspected to comprisebreast cancer, wherein if the polypeptide is not detected it isindicative of breast cancer in the sample.
 95. The method of claim 94,wherein the detecting comprises contacting the biological sample with anantibody that is immunologically reactive with the polypeptide.
 96. Amethod of producing an antibody that is immunoreactive with thepolypeptide of any one of claims 89 to 92, the method comprising: (a)immunizing a non-human animal with the polypeptide of any one of claims89 to 92, or a fragment thereof; and (b) recovering an antibody from thenon-human animal.
 97. A method of producing an antibody immunoreactivewith the polypeptide of any one of claims 89 to 92, the methodcomprising: (a) transfecting a recombinant host cell with a nucleic acidsequence encoding the polypeptide of any one of claims 89 to 92; (b)culturing the host cell under conditions sufficient for expression ofthe polypeptide; (c) recovering the polypeptide; and (d) preparing anantibody to the polypeptide.